abiâ„¢ 3948 reference manual - life technologies

ABI

™

3948

Nucleic Acid Synthesis and Purification System

Reference Manual

© Copyright 2001, 2011 Applied Biosystems. All rights reserved.

For Research Use Only. Not for use in diagnostic procedures.

ABI P

RISM

and the ABI P

RISM

design, Applied Biosystems, FastPhoramidite, ONESTEP, OPC, Phosphalink, and POLYPORE are registered trademarksof Applera Corporation or its subsidiaries in the U.S. and certain other countries.

ABI, CATALYST, LV40, and PDQ are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

All other trademarks are the sole property of their respective owners.

Contents

iii

1 Introduction to the Reference Manual. . . . . . . . . . . . . . . . . . . . . 1-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

Using This Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

User Bulletins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Key Terms Defined . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

2 System Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

Section 1 - General Description of Processes, System Control, and Hardware . . . . . . . . . 2-2

Basic Instrument Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3

General Description of Instrument Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4

System Control and Sensing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

Hardware Description Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

Section 2 - More Details of System Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8

OneStep Column and Column Turntable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-9

Jaw Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-11

Pressure Regulation and Control (PRC) Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12

Synthesis Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13

Cleavage Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-15

Deprotection Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-16

Purification Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18

Quantitation Module. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-20

Sample Collection Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-21

3 Automated DNA Synthesis Chemistry . . . . . . . . . . . . . . . . . . . . . 3-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

Section 1 – DNA Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

DNA Synthesis Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

Overview of the Phosphoramidite Method of Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6

Detritylation Chemistry Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

Coupling Chemistry Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-11

Capping Chemistry Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-13

Oxidation Chemistry Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15

DNA Cleavage and Deprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18

iv

Section 2 – Oligonucleotide Purification, Quantitation, and Storage . . . . . . . . . . . . . . . 3-19

DNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20

Overview of Oligonucleotide Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-22

Measurement of ODU and Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-23

Storage of the Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-27

Alternative Chemistries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-28

4 Overview of Software Commands . . . . . . . . . . . . . . . . . . . . . . . . 4-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

Section 1 – File and Edit Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2

Overview of File Menu Commands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

New Synthesis Order Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5

Open Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Open Synthesizer Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10

Other File Menu Commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13

Overview of Edit Menu Commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-17

Section 2 – Synthesizer and Window Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-20

Overview of Synthesizer Commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-21

Abort Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-22

Interrupt Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-22

Resume Command. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-22

Pause After Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23

Synchronize Clocks Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-24

Change Password Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-25

Change Name Command. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-26

Instrument Preferences Command. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-27

Window Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-30

Section 3 – RunFiles, Sample Labeling, Synthesis Order and Multi-Order Files . . . . . 4-31

RunFiles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-32

Sample Labeling Feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-33

Label View Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-36

About Synthesis Orders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-38

Multi-Order Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-41

5 Communication View and Operational Views . . . . . . . . . . . . . . . 5-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1

Section 1 - General Information about Operational Synthesizer Views . . . . . . . . . . . . . . 5-2

About the Synthesizer Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4

Communication View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6

Run Setup View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7

Run Protocol View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9

v

Monitor Chemistry View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11

Monitor Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15

Liquid Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16

Monitor Run View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-19

Section 2 - Using the Run Setup View and Extending a Run . . . . . . . . . . . . . . . . . . . . . . 5-20

Importing Synthesis Orders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-21

Selecting Orders for the Next Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-23

Synthesis Order Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-24

Assigning Chemistry and AutoSorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-25

Bottle Usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-27

Using the Load Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-28

Extending a Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-30

6 Synthesizer Views Supporting Operation . . . . . . . . . . . . . . . . . . . 6-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

Section 1 – Non-Cycle Operational Support Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2

Manual Control View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3

Power Fail View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6

Instrument Test View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-7

B+ Tet Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8

Reagent Utilization Table View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9

Section 2 – Editing Cycle and Procedure Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11

Edit Cycle and Procedure Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-12

Edit Synthesis Cycle View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-17

Edit Cleavage Cycle View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-18

Edit Purification Cycle View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-19

Edit Begin Procedure View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20

Edit End Procedure View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21

Edit Bottle Procedure View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22

Misc (Miscellaneous) Procedures View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23

A Valves and Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1

In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1

Information on ABI 3948 System Chemistry Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . A-3

Valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5

Overview of Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7

Valve Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-10

Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-11

Valve Functions by Functional Category . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-15

Non-Valve Hardware Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-23

vi

B ABI 3948 System Special Functions . . . . . . . . . . . . . . . . . . . . . . B-1

In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1

Overview of Special Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2

Listing of Special Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-5

C Cycles, Procedures, and System Messages . . . . . . . . . . . . . . . . . C-1

In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

3948 Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2

The Standard Synthesis Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-3

The Standard Cleavage/Deprotection Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-9

The Standard Purification Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-18

Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-32

System Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-38

D 3948 System Function List and Other Cycles . . . . . . . . . . . . . . . D-1

In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-1

ABI 3948 System Function List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

The Purification Dye Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-39

The Purification Biotin Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-48

E Instrument Plumbing Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . E-1

F Limited Warranty Statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1

Index

Introduction to the Reference Manual 1-1

Introduction to the Reference Manual 1

In This Chapter

User and ReferenceManuals

The

ABI

3948 System Reference Manual

is one of two manuals in the document set supporting the ABI

™

3948 Nucleic Acid Synthesis and Purification System. The first manual in the set, the

ABI

3948

System User’s Manual

, is intended for daily operation, while this reference manual presents detailed information needed to fully understand, use, and maintain the instrument.

Topics Covered

This chapter contains the following topics:

Topic See page

Using This Manual 1-2

Contents of the Manual 1-2

User Bulletins 1-4

Purpose of User Bulletins 1-4

Key Terms Defined 1-4

Table of Key Terms 1-4

1

1-2 Introduction to the Reference Manual

Using This Manual

Contents of theManual

This manual contains five chapters and several appendices.

Chapter/Appendix Title Types of Information

1 Introduction to the Reference Manual/ Instrument

�

Purposes of User and Reference manuals

�

Information about safety and user bulletins

�

Description of the ABI 3948 System

2 System Description

�

Overview of how the system is controlled

�

Descriptions of the six modules:

– Synthesis

– Cleavage

– Deprotection

– Purification

– Quantitation

– Sample Collection

3 Chemistry for Automated DNA Chemistry

�

DNA synthesis overview

�

Description of Cleavage and Deprotection

�

Quantitation of the Oligonucleotide

�

Purification

�

Storage of the Oligonucleotide

4 Overview of System Commands

Detailed command information covering the following menus:

�

File menu

�

Open Synthesizer menu

�

Edit menu

�

Synthesizer menu

�

Window menu

5 Communication View and Operational Views

Detailed information covering the following:

�

Synthesizer Window

�

Communication view

�

Run Setup view

�

Run Protocol view

�

Monitor Chemistry view

�

Monitor Instrument view

�

Monitor Run view

Introduction to the Reference Manual 1-3

6 Other Synthesizer Views

Detailed information covering the following:

�

MISC (Miscellaneous) Procedures view

�

Manual Control view

�

Power Fail view

�

Instrument Test view

�

B+Tet Calibration view

�

Reagent Utilization Table view

�

Editing Cycle and Procedure views

A Valves and Functions

�

Default Contents of the B+ Tet Calibration View

�

Default Contents of the Reagent Utilization View

B Cycles, Procedures, and System Messages

Detailed information on the following:

�

Standard Synthesis Cycle

�

Standard Cleavage/Deprotection Cycle

�

Standard Purification Cycle

�

Procedures

�

System Messages

C 3948 System Function List and Other Cycles

�

3948 System Function List

�

Purification Dye Cycle

�

Purification Biotin Cycle

Chapter/Appendix Title Types of Information

1-4 Introduction to the Reference Manual

User Bulletins

Purpose of UserBulletins

User Bulletins (UBs) contain technical information that is essential to ABI 3948 instrument operation and related laboratory techniques. UBs are the quickest way to ensure that you have current information. They are produced periodically and mailed to you as they become available.

Please read the UBs before operating your ABI 3948 instrument. Current UBs are found under their own tab at the end of the

ABI 3948 Instrument Reference Manua

l.

Key Terms Defined

Table of Key Terms

The following table lists the key terms needed for operation of the 3948.:

Term Definition

3948Control Views

Protocol Contains the three chemistry cycles needed to produce an oligonucleotide:

�

Synthesis cycle

�

Cleavage cycle

�

Purification cycle

When new chemistry cycles are available, you can assign them to a new protocol using the Run Protocol view. For more information on creating a new protocol with your own cycles, see Chapter 6 of this manual.

Begin and End Procedures

These procedures, chosen during Run Setup, are run before and after oligonucleotide production. If you create a new protocol with your own cycles, you may need to create revised versions of these procedures (see Chapter 6 for information on revising these procedures).

Commands

Abort Immediately terminates execution of a run or Manual Control action in progress.

Interrupt Halts the instrument at the first safe step for all active chemistries (synthesis, cleavage, purification, and procedures).

There are two ways to initiate an interrupt:

� Choose Interrupt from the Synthesizer menu

� Press the Interrupt button on the ABI 3948 Synthesizer Front Panel

If you use Interrupt to halt the instrument operation, use the Resume command from the Synthesizer menu to restart the instrument

Pause After Halts the instrument after the designated synthesis, allowing the user to extend the run or do a bottle change if necessary.

To initiate a Pause After, choose Pause After from the Synthesizer menu.

System Description 2-1

System Description 2

In This Chapter

Topics Covered This chapter provides a general description of instrument chemistry processes and hardware.

The chapter contains two sections:

Topic See page

General Description of Processes, System Control, and Hardware 2-2

More Details of System Hardware 2-8

2

2-2 System Description

Section 1 - General Description of Processes, System Control, and Hardware

Topics Covered This section provides a general description of instrument Chemistry and other processes, system control and sensing, and an overview of instrument hardware.

The section covers the following topics:

Basic Instrument Features 2-3

Automatic Oligonucleotide Production 2-3

Phosphoramidite Method of Synthesis 2-3

Pressure-Driven Chemical Delivery 2-3

Macintosh 3948Control Software 2-3

General Description of Instrument Chemistry 2-4

Automation of Chemistry Processes 2-4

The First Four Chemistry Processes 2-4

Quantitation 2-5

Sample Collection 2-5

System Control and Sensing 2-6

Introduction 2-6

Types of Storage 2-6

Electromechanical and Electrical Drivers 2-6

Gas Pressure Sensors/Sensor Drivers 2-6

Liquid Sensors 2-6

Hardware Description Overview 2-7

Types of Hardware Described 2-7


Basic Instrument Features

AutomaticOligonucleotide

Production

The ABI 3948 Nucleic Acid Synthesis and Purification system completely automatesthe entire process of oligonucleotide production: synthesis, cleavage, deprotection,purification, quantitation, and sample collection. When used as a system utilizing ABIreagents and columns, this instrument produces high quality synthetic DNA whileminimizing synthesis time and cost.

PhosphoramiditeMethod of Synthesis

The phosphoramidite method of oligonucleotide synthesis is used because of itsinherently high coupling efficiency and the stability of the starting materials. The 3´terminal nucleoside attached to a solid support, which is contained within a disposablecolumn (the OneStep™ column). Nucleoside bases are added one at a time to thesupport-bound DNA chain until the sequence is fully synthesized. Solid supportsynthesis allows excess reagents to be removed by filtration and eliminates the needfor purification between base additions.

Pressure-DrivenChemical Delivery

Applied Biosystems synthesizers use a pressure driven chemical delivery system todeliver reagents and solvents to a reaction column chamber (OneStep column).Reagent and solvent deliveries also rely on our zero-dead volume valves whichincrease reliability, eliminate cross-contamination and reduce cycle costs.

ABI 3948 SystemSoftware for

Macintosh

You can program cycles, functions, and procedures for use in the synthesizer from theABI 3948 system software for Macintosh computer. Once you download chemistryprotocols, an internal controller/driver within the synthesizer exercises real-timecontrol of the instrument. The ABI 3948 instrument can run preprogrammed protocolsor you can create customized cycles. The Macintosh software is also used to fill outthe Synthesis Orders used as sequence input for the instrument.


General Description of Instrument Chemistry

Automation ofChemistry Processes

The ABI™ 3948 DNA Synthesis and Purification system automates four processes of DNA chemistry (synthesis, cleavage, deprotection, and purification), and then quantitates and automatically collects the DNA product.

The synthesis, cleavage, and purification processes are implemented in three modules that operate concurrently to provide the high throughput capacity of the instrument—as many as 48 twenty-mer oligonucleotides per day of operation.

The First FourChemistry Processes

Three of the four chemistry processes (synthesis, cleavage, and purification) take place within the OneStep™ column as it moves between the three modules. The fourth process, deprotection, takes place outside the OneStep column in the deprotection coils between cleavage and purification.

I

Module Process

Synthesis module The first process occurs in the column while it is positioned at the Synthesis module. The OneStep column contains the 3´-terminal nucleoside, covalently attached to support material. The DNA chain is built by adding one base at a time to the support-bound nucleoside.

IMPORTANT You can synthesize crude oligonucleotides with the 5´ trityl group either on or off; you must synthesize purified oligonucleotides with the 5´ trityl group on.

Cleavage module During the second process of automated oligonucleotide production, the OneStep column is positioned at the Cleavage module and processed as follows:

a. In the Cleavage module a delivery of concentrated aqueous ammonium hydroxide cleaves the oligonucleotide from the support.

b. The DNA product in the ammonium hydroxide solution is then transferred to the Deprotection module for processing outside the column.

Deprotection module In the Deprotection module, protecting groups are removed from the oligonucleotide as the third process by heating the solution containing the oligonucleotide for one hour at 65 °C.

Purification module During the fourth DNA chemistry process, purification, the desired, full-length oligonucleotide is separated from failure sequences and other impurities at the Purification module.

Purification is performed as follows:

a. The deprotected product is passed in ammonium hydroxide through the support in the original OneStep column.

b. The tritylated, full-length oligonucleotide binds to the support in the column while detritylated failure sequences and other impurities are washed away.

c. Following detritylation, the DNA products are eluted from the OneStep column, using a solution of 20% acetonitrile in water


Quantitation In the fifth production process, the purified oligonucleotides are quantitated using a UV (ultraviolet) light detection system as follows.

� The product, in a 20% acetonitrile solution, passes through the detector as it is delivered to the sample collection module.

� The absorbance is reported in ODUs (Optical Density Units) and picomoles to indicate quantity.

Sample Collection Automated oligonucleotide production concludes with the collection of the DNA sample in the sample collection module:

� The sample collection tray contains 48 sample vials to allow unattended collection of 48 DNA samples.

� A septum sheet under the OligoRack™ cover minimizes evaporation from and contamination of the sample vials.

� Each of the sample vials, covered by a septum, holds the 1-mL DNA sample volume from a OneStep column obtained in either the deprotection or purification module. The vial septum is chemically inert to both 20% acetonitrile/H2O and ammonium hydroxide.


System Control and Sensing

Introduction The ABI™ 3948 DNA Synthesis and Purification System is controlled from a computer with the Macintosh-compatible ABI 3948 instrument operating software. A user programs the system through the computer which interacts with embedded software on the instrument.

Types of Storage Several types of memory storage are available on the instrument:

� The embedded software code allows real-time control via the Macintosh® computer.

� The 3948 boot ROM holds instrument-unique data such as the instrument serial number.

� Battery backed-up memory stores critical data, such as power-failure recovery information and any data that is not transferred to the Macintosh.

� Temporary memory is reserved for the proper and reliable operation of the instrument. It is possible to increase synthesizer memory to accommodate future upgrades.

Electromechanicaland Electrical

Drivers

The instrument contains a number of electromechanical drivers that control the process of oligonucleotide production:

� One of these, the driver for the OneStep column turntable, properly positions the OneStep columns in the jaw mechanisms.

� Another driver, for the jaw mechanism, engages and disengages the jaw module to ensure a leak-tight seal.

� Other driver mechanisms include: the deprotection heater driver, the sample collection driver, and valve drivers.

Gas PressureSensors/Sensor

Drivers

Gas pressure sensors ensure accurate pressure-regulated delivery rates. In addition to regulation of gas and liquid flows, sensor drivers monitor the following instrument operations:

� Maintenance of the battery supported memory

� Positioning and movement of the OneStep column turntable and the jaw mechanism

� Regulation of the deprotection system temperature

� Sample collection

� Regulation and usage of the pressure management system that provides automated leak test capability

� Valve driver power usage and load/no-load testing to detect solenoid shorts and disconnections

Liquid Sensors The instrument also has liquid sensors to ensure that adequate amounts of reagents are available and properly delivered during the run. Liquid sensors are shown in the figure on page 2-16.


Hardware Description Overview

Types of HardwareDescribed

The following hardware is described in this chapter:

� OneStep™ Column and Column Turntable

Four types of OneStep columns are used. One for each of the starting monomers. The turntable is used to hold the columns and move them between the three chemistry jaw mechanisms (Synthesis, Cleavage, and Purification).

� Jaw Mechanism

This is the device used to clamp on the top and bottom of OneStep columns and provide flow paths for chemistry.

� Pressure Regulation and Control Module (PRC)

This module regulates and controls various pressures in the system.

� Synthesis Module

This module consists of the Synthesis jaw mechanism and associated plumbing.

� Cleavage Module

This module consists of the Cleavage jaw mechanism and associated plumbing.

� Deprotection Module

This module consists of deprotection coils and associated plumbing.

� Purification Module

This module consists of the Purification jaw mechanism and associated plumbing.

� Quantitation Module

This module consists of the UV detector and associated plumbing.

� Sample Collection Module

This module consists of the following three components:

– A mechanism and tray to hold the OligoRack/red rack and waste bottles.

The mechanism moves the tray to position tubes under the needle delivery system and extend the tray so that individual tubes can be removed.

– An OligoRack or optional red rack to hold 1.2-mL tubes.

– A needle delivery system to individually delivery synthesized or full length oligonucleotides to assigned positions.


Section 2 - More Details of System Hardware

This section provides general information on system hardware.

OneStep Column and Column Turntable 2-9

OneStep Columns and Column Turntable 2-9

OneStep Column Turntable 2-10

Jaw Mechanism 2-11

Uses of the Three Jaw Mechanisms 2-11

Pressure Regulation and Control (PRC) 2-12

Characteristics of Module 2-12

Synthesis Module 2-13

Location on the Plumbing Diagram 2-13

The Process of Synthesis 2-14

Cleavage Module 2-15


The Process of Cleavage 2-15

Deprotection Module 2-16

Deprotection Module Components 2-16


The Process of Deprotection 2-17

Purification Module 2-18


The Process of Purification 2-19

Quantitation Module 2-20

Description of UV Detection System 2-20

Sample Collection Module 2-21

Three Main Components 2-21

Two Types of Racks 2-22

Waste Containers 2-22


OneStep Column and Column Turntable

OneStep Column The OneStep column, shown in orthogonal and cross-sectional views below, contains a proprietary support composed of a highly cross-linked polystyrene with the pore and particle sizes appropriate for both synthesis and purification.

The OneStep column has these characteristics:

� Color coded to identify the starting monomer (A=green, G=yellow, C=red, and T=blue)

� Accommodates 40 nmol of pure DNA product

� Easy to load and remove from the column tray

� Chemically inert to all chemicals used on the ABI 3948 and is designed to provide a leak-free seal with the synthesis, cleavage, and purification modules

Orthogonal view Cross-sectional view

GR

02

11

•Mo

de

l 39

48

Re

fern

ce M

an

ua

l�O

rtho

gra

ph

ic view

of C

olu

mn

= Filter Material= Synthesis PowderSynthesis support

Filter (frit) material (pore size)


OneStep ColumnTurntable

The OneStep column turntable, shown below, does the following:

� Positions the columns in the synthesis, cleavage, and purification modules to ensure that the jaw mechanisms at these modules form a leak-free seal.

� Holds 48 columns and is easy to load.

� Precisely indexes the position of each column on the turntable for software control of each step of chemical delivery.

Note For clarity, the turntable in the figure is shown pulled away from the three jaw mechanisms with which it is associated. As the turntable rotates, OneStep columns in all 48 positions are presented in turn to the three jaw mechanisms for reagent deliveries.


Jaw Mechanism

Uses of the ThreeJaw Mechanisms

The jaw mechanism, shown below, is used in conjunction with the OneStep columns and turntable to provide a leak-tight seal for the reagent/gas delivery system.

Three jaws, one for each of the three chemistry modules (synthesis, cleavage, and purification), operate independently so that you can perform all three chemistries concurrently. All wetted components used in the mechanism are chemically inert to all reagents used for synthesis, cleavage and purification.


Pressure Regulation and Control (PRC) Module

Characteristics ofModule

The PRC board assembly function:

� Provides accurate gas delivery, regulates pressure to ensure sufficient volumes of reagents, and closes down delivery at shut off.

� Is entirely controlled via firmware and software.

� Has 10 individually selectable valves with 3 outlet ports for each valve.

� Individually controls the pressure settings of each valve via software. The pressure can be variably set from 0 psig (off) and up to 12 psig.

Note All used outlet port have a check valve and un-used ports are plugged.

� Has the following pressure transducers:

– Ten low pressure transducers to enable monitoring of pressure in the “Monitor Instrument” View.

– One pressure transducer to monitor inlet gas pressure.


Synthesis Module

Location on thePlumbing Diagram

The Synthesis module is a station on the column turntable where synthesis is performed on up to three reaction cartridges at a time. The portion of the plumbing diagram containing the Synthesis module is shown below. Liquid sensors, like those indicated in the plumbing diagram, are shown on page 2-16. For the full instrument plumbing diagram, see Appendix E.


The Process ofSynthesis

Synthesis is performed by delivering phosphoramidites and reagents through a OneStep column while the column is clamped firmly in a jaw mechanism to ensure leak free delivery:

� Controlled quantities of reagents are supplied step-by-step to the module from reagent bottles by a system of valve blocks using positive-lift valves and gas/ liquid sensors.

� Phosphoramidites are supplied from 2-g bottles and reagents are supplied in the following bottle sizes:

– TCA (2L), ACN (4 L)

– Iodine (450 mL)

– Acetic Anhydride (450 mL)

– Tetrazole (450 mL)

– NMI (450 mL)

Reagent volumes correspond roughly to the rate at which they are consumed during synthesis.


Cleavage Module


The Cleavage module is a station on the column turntable where cleavage is performed on up to three columns at a time during the cleavage cycle. The portion of the plumbing diagram containing the Cleavage module is shown below. Liquid sensors, like those indicated in the plumbing diagram, are shown in the figure on page 2-16. For the full instrument plumbing diagram, see Appendix E.

The Process ofCleavage

Cleavage is performed by delivery of concentrated ammonium hydroxide through a OneStep column while the column is clamped firmly in a jaw mechanism to ensure leak free delivery.


Deprotection Module

DeprotectionModule Components

Unlike the Synthesis, Cleavage, and Purification processes, which are performed within the OneStep columns, deprotection is performed on the product in solution outside of these columns. The components of the Deprotection module are shown below.


The portion of the plumbing diagram containing the Deprotection module is shown below. For the full instrument plumbing diagram, see Appendix E.

Heater housing block

Valve solenoids

Valve blocks

Liquid sensors

Deprotection coils


The Process ofDeprotection

Deprotection includes the following:

� Eluting the product in solution from the column in the Cleavage module and transferring it to one of three coils in the Deprotection module heater (see figure above).

Note Liquid sensors monitor the transfer of solution to the coils.

� Removing the remaining protecting groups by heating the product in solution for a programmed period of time at 65 °C.

Note All wetted components used in the module are chemically inert to ammonium hydroxide.


Purification Module


Like Synthesis and Cleavage, Purification is performed within the OneStep columns in this module. Liquid sensors, like those indicated in the plumbing diagram, are shown in the figure on page 2-16. The portion of the plumbing diagram containing the Purification module is shown below. For the full instrument plumbing diagram, see Appendix E.


The Process ofPurification

Purification is performed using a trity-selective purification chemistry to separate the desired full-length olignonucleotides from failure sequences and impurities:

� The deprotected product, dissolved in the cleavage solution (NH4OH), passes through the columns which contain a purification support.

� The tritylated full-length oligonucleotide binds to the support while detritylated failure sequences and other impurities are washed away.

� The 5´-terminal trityl group, which was not removed at the conclusion of synthesis, is removed with 3% TFA.

� The oligonucleotide product is eluted from the column with a solution of 20% acetonitrile in water.

.


Quantitation Module

Description of UVDetection System

Oligonucleotides are quantitated in a 20% acetonitrile solution using a UV detection system:

� A low-pressure mercury lamp (P/N 100750) in the UV detector, shown below, provides a UV-light source at 254.5 nm.

� As each oligonucleotide passes out of the purification module, through the cuvette on its way to the sample collection system, current is measured (mV) and then converted into optical density units and picomoles.


Sample Collection Module

Three MainComponents

Samples are collected in the ABI™ 3948 Nucleic Acid Synthesis and Purification System by a sample collector system with three components:

� An OligoRack (red rack) or white rack with 48 positions for 1.2-mL tubes

� A needle delivery system to individually deliver synthesized or full length oligonucleotides to assigned positions;

� A mechanism that moves the tray so that you can properly remove either an entire rack or individual tubes from each of the 48 positions (see figure below).

Note This is a rear three-quarter view of the module. The sample collection tray, which holds an OligoRack, is shown extended in the figure and by itself below. At the top of the figure is the sample collector needle assembly.


Note The sample collector tray is used to move an inserted rack into and out of the Sample Collection module. The two circular openings at the rear are used to hold waste bottles.

Two Types of Racks The two types of racks have the following configurations:

� The 4X12 configuration rack that comes with the instrument, the white rack, is not disposable and cannot be purchased separately (the screw top tubes in the rack are removed for customers).

� The optional red rack may be purchased to enable an entire rack to be shipped to a single customer. This type of rack has an 8X6 configuration with included vials and press tops.

The two racks measure 8.5 x 12.7 x 5.5 cm and are shown in the Sample Collector tray below:

Note The red rack contains 6 rows of 8 tubes alternating with 6 rows of tube caps.

Each of the two types of racks contains 48 sample positions and has the following characteristics:

� One corner of each rack cover is beveled. When positioned correctly on the sample collection tray, the beveled corner faces the inside of the instrument.

� Each rack cover is lined with a septum to minimize evaporation and contamination.

� The tubes and all wetted parts of the system are chemically inert to the 20% acetonitrile solution used to elute oligonucleotides and to concentrated ammonium hydroxide.

Waste Containers Two empty bottles serve as waste collection vials as follows:

� Between samples, the collection needle is rinsed.

� The rinses are delivered to one of the two waste bottles.

� You must empty these waste bottles after each 3948 run. Bottles are located in the circular opening shown in the figure above.

IMPORTANT Refer to the Chemical Safety section of the ABI™ 3938 DNA Synthesizer Site Preparation and Safety Guide for a description of the contents of this collected waste.

key

Position #1

Position #48

Rack with 4 x12 formatOptional Red Rack with 6 x 8

Position #1

(white - uses screw top tubes) format - uses press top vials

10-mL sample collectorwaste bottles positions

Position #48

Automated DNA Synthesis Chemistry 3-1

Automated DNA Synthesis Chemistry 3

In This Chapter

Topics Covered This chapter presents a description of DNA synthesis chemistry. In addition, it contains an overview of oligonucleotide analysis and purification procedures, and information about quantifying and storing oligonucleotides.

This chapter contains two sections:

Section See page

DNA Chemistry 3-2

Oligonucleotide Quantitation, Purification, and Storage 3-19

3

3-2 Automated DNA Synthesis Chemistry

Section 1 – DNA Chemistry

Topics Covered This section describes how DNA synthesis chemistry is implemented on the solid support within the OneStep™ column on the ABI™ 3948 Nucleic Acid Synthesis and Purification System.


Topic See page

DNA Synthesis Overview 3-3

DNA Synthesis Process 3-3

DNA Synthesis Cycle 3-3

Solid Support 3-3

Completion of the Synthesis Cycle 3-5

Overview of the Phosphoramidite Method of Synthesis 3-6

Four Chemistry Reactions 3-6

Chemistry of Choice 3-7

Phosphoramidite Nucleotides 3-7

Functional Groups 3-8

Detritylation Chemistry Reactions 3-9

Detritylation Process 3-9

Depurination 3-9

Coupling Chemistry Reactions 3-11

Washing and Drying of Support 3-11

Simultaneous Delivery of Phosphoramidites and Tetrazole 3-11

Capping Chemistry Reactions 3-13

Why Capping is Needed 3-13

Capping Process 3-13

Oxidation Chemistry Reactions 3-15

Oxidation Reaction 3-15

Importance of Oxidation After Capping 3-15

DNA Cleavage and Deprotection 3-16

Introduction 3-16

Cleavage 3-16

Phosphate Deprotection 3-16

Base Deprotection 3-17

References 3-18


DNA Synthesis Overview

DNA SynthesisProcess

In DNA synthesis, synthesis proceeds as follows:

DNA Synthesis Cycle Each cycle of nucleotide addition consists of four steps, which differ depending on the desired structure of the final product.

� Detritylation

� Coupling

� Capping

� Oxidation

These reaction steps are repeated in the same order until all nucleotides in the sequences have been added. The operator of the DNA synthesizer defines the desired sequence and length of the final product.1 Following synthesis, the oligonucleotide is cleaved and deprotected from the solid support.

Solid Support The ABI™ 3948 Nucleic Acid Synthesis and Purification System uses a solid-phase synthesis chemistry with a highly cross-linked polystyrene support and attaches the nucleotide 3´-hydroxyl to the support with an aminomethyl linker.

The following support characteristics are described in this subsection:

� Solid Phase Synthesis Chemistry

� Highly Cross-linked Polystyrene Support

� Attachment of nucleotide 3´-hydroxyl to Support by Linker

Solid Phase Synthesis Chemistry

The ABI 3948 DNA Synthesizer uses solid phase chemistry:

Stage Description

1 A reactive 3´ phosphorus group of one nucleotide (in the form of a monomer in solution) is coupled to the 5´ hydroxyl of a nucleotide immobilized on a solid support.

2 An internucleotide linkage forms as a result of the coupling.

3 Three more chemical reactions follow to prepare the growing chain of DNA for the next coupling.

In each DNA synthesis cycle, one nucleotide monomer is added to the chain.

Stage Description

1 Before beginning a synthesis, one of the support-bound nucleotides (A, C, G, or T) contained within a OneStep™ column, is placed on the instrument.

2 As synthesis proceeds:

� All reagents and solvents flow through the support, which is contained within a unique synthesis/ purification column

� The growing DNA chain remains covalently attached to the insoluble synthesis support within the column.


Highly Cross-linked Polystyrene Support

The support used for DNA synthesis/purification is a highly cross-linked polystyrene with the following characteristics during major phases of chemistry:

Attachment of Nucleotide 3´-hydroxyl to Support by Linker

As shown in the figure below, polystyrene has an aminomethyl linker attached to its surface. This type of linker enables the support characteristics listed in the table which follows the figure.

Note The DMT-protected nucleotide is attached to the polystyrene support (B = Base, A,G,C,T).

During… The support…

Synthesis produces coupling efficiencies of about 98% and higher, as measured by the trityl cation assay.

Purification is also used as a purification media for the DNA.

Note The support can be used for purification because the polystyrene used is a porous, non-swelling bead which exhibits hydrophobic properties.

SupportCharacteristic Description

Inert surface Side reactions occur less frequently because the surface of polystyrene is inert.

Easy derivatization

The supports are derivatized by covalently attaching the 3´-hydroxyl of the nucleotide to the linker via a succinate ester bond, which is base-labile and allows for removal of the DNA from the support with ammonia.

Quantitative cleavage

After synthesis is complete, the oligonucleotide is quantitatively cleaved, leaving a free 3´-hydroxyl.

BP

= Abz

G

C

T

bzdmf

O BDMTO

OCCH2CH2CNHCH2

O O

polystyrene

P


Completion of theSynthesis Cycle

After an individual base undergoes oxidation, when other base additions are to follow, the synthesis cycle is completed as follows:

Note When making crude DNA with 5´ labels that do not have a terminal DMT (such as a dye label), always use the “Crude-DMT on” option in order to prevent excess exposure of the label to TCA. Always consult the instructions provided with your particular label for the correct handling procedure.

Phases Description

DMT removal

After the oxidation of a particular base, the dimethoxytrityl group is removed with trichloroacetic acid to prepare for the next coupling.

Coupling/Capping/Oxidation

The previous step and these steps are repeated until chain elongation is complete.

Note At this point, the oligonucleotide is still bound to the support with protecting groups on the phosphates and the exocyclic amines of the bases A, G, and C

Concluding phase

One of the following three options is chosen when setting up for the run, to control the last phase:

If… Then

the oligonucleotide is be be purified

choose “Purify Oligo” on the Synthesis Order for the sequence.

Note When synthesis is complete, the DMT group is still attached to the 5´- terminus enabling it to act as a hydrophobic handle for purification.

the oligonucleotide is not to be purified

choice one of the following Crude options:

� “Crude - DMT on” if the DMT group is not to be removed from the sequence.

� “Crude - DMT off” if the DMT group is to be removed.


Overview of the Phosphoramidite Method of Synthesis

Four ChemistryReactions

A synthesis cycle is depicted below using the phosphoramidite method of oligonucleotide synthesis. As the figure demonstrates, the synthesis cycle consists of four chemical reactions:

� Detritylation

� Coupling

� Capping

� Oxidation

O

O

BDMTO

P OCH2CH2CNiPr2N

O

O

BDMTO

P OCH2CH2CN

O O

O

B

nucleosidesupport-bound

Phosphoramidite nucleoside

O

O

BDMTO

P OCH2CH2CNO

O O

O

B

O

O

BHO

O

O

BDMTO

COUPLING

CAPPING

O

O

BCH3CO

O

OXIDATION

DETRITYLATION

P

P

P

P

P

P

P Add Monomer

Solid Support

CAPPING

nucleotide Phosphoramidite


Chemistry of Choice The phosphoramidite method of oligonucleotide synthesis is the chemistry of choice for most laboratories because it offers efficient, rapid coupling and stable reagents2.

Efficient, rapid coupling is enhanced by the following phosphoramidite chemistry characteristics:

PhosphoramiditeNucleotides

Structures and Molecular Weights

Phosphoramidites are chemically-modified nucleotides that are the building blocks for synthesized oligonucleotides.

Note This figure shows the structures and molecular weights of standard cyanoethylphos- phoramidite monomers. Exocyclic adenine (A) and cytosine (C) are protected with the benzoyl group; the exocylic amine of guanine (G) is protected by a dimethylformamidine group (dmf). Thymine (T) does not need a protecting group.

Characteristic Description

Method of derivitization

The solid support upon which synthesis begins is derivatized with the nucleotide which becomes the 3´-hydroxyl end of the oligonucleotide. This support material consists of beads composed of a polymer.

Support particle and pore sizes

Support particle and pore sizes are optimized for liquid transfer and mechanical strength.

Removal of excess reagents

Excess reagents in the liquid phase can be removed by filtration and washing3, eliminating the need for purification steps between cycles.

Guanine - dimethylformamidineAdenine - benzoyl protected

Cytosine - benzoyl protectedThymine

MW 857.95 protectedMW 824.92 C43H53N8O7P

MW 744.83C40H49N4O8P

MW 833.93C46H52N5O8P

C47H52N7O7P


Functional Groups Cyanoethyl phosphoramidite nucleotides have four functional groups

Functional Group Description

The diisopropylamino on a 3´ trivalent phosphorous moiety4

This group stabilizes the phosphoramidite and is made highly reactive by the activator, tetrazole.

The cyanoethyl protecting group on the 3´ phosphorous moiety5

This group prevents side reactions and aids in solubility of phosphoramidites.

� Upon completion of the synthesis, it is removed with ammonium hydroxide.

� In deprotection, ammonia acts as a base to remove a proton on the methylene group bearing the nitrile group.

This anion is formed only in low concentration, but rapidly fragments by a beta-elimination reaction to form acrylonitrile and the deprotected internucleotide phosphodiester group. Acrylonitrile then reacts irreversibly with ammonia to form 3-aminopropionitrile, an inert compound.

The dimethoxytrityl (DMT, trityl) protecting group on the 5´ hydroxyl

This group is removed during each detritylation step leaving a reactive 5´ hydroxyl available for coupling an incoming phosphoramidite.

The protecting groups on the exocyclic amines of Abz, Cbz, Gdmf (bz = benzoyl, dmf = dimethylformadine group)

These groups prevents side reactions and are removed upon completion of the synthesis with ammonia. Thymidine is not protected, because it does not contain an exocyclic amine moiety, and therefore is unreactive.


Detritylation Chemistry Reactions

Detritylation Process Detritylation, the first step of oligonucleotide synthesis cycle, is treatment of the derivatized solid support with the protic acid, trichloroacetic acid (TCA) in dichloromethane, to remove the acid-labile, DMT-protecting group (as shown in the figure below). This yields a reactive 5´ hydroxyl which can couple with a nucleotide phosphoramidite monomer during the following coupling reaction.

Detritylation proceeds as follows:

Depurination Purines Susceptible to Depurination

Trichloroacetic acid is a very effective protic acid detritylating agent. However, in high concentrations of protic acids, the amine-protected purines (Abz and Gdmf) are susceptible to depurination (removal of the purine from its sugar) via the following pathway.6

Initial protonation at N-7 of the purine ring increases the lability of the ribose 1´-purine N-9 bond. Cleavage of adenine and guanine bases yields a 1´ hemiacetal ribose ring, the result of depurination. Then, during ammonium hydroxide treatment, Cleavage occurs at the internucleotide bonds on the 3´-hydroxyl side of the apurinic deoxyribose. (This is similar in effect to the chemistry of Maxam-Gilbert sequencing.)

Differential Exposure

Each purine in the oligonucleotide chain is exposed to acid at each detritylation step. Purines near the 3´- end will have the longest cumulative exposure time and a greater chance for depurination.

Stage Description

1 Immediately before detritylation, the support is washed with acetonitrile to eliminate traces of the preceding reagent.

Note Detritylation under anhydrous conditions is a reversible reaction. The trityl cation is highly reactive and can re-tritylate any reactive nucleophile.

2 Detritylation is driven to completion by the removal of the trityl cation from the synthesis/purification column.

The trityl cation is eluted by several TCA deliveries.

3 The final detritylation step is a trityl flush in which argon passes through the column from bottom to top and pushes the liquid to a halogenated waste reservoir.

Any residual TCA is removed by an acetonitrile wash.


Note It has also been reported that a 5´ terminal purine is more susceptible to depurination than an internucleotide purine.7

Quantity of DNA Cleavage Products

The quantity of DNA cleavage products generated by apurinic ammoniolysis is usually insignificant and should not affect your product significantly.

Depurination is usually neither detectable nor significant when using ABD reagents and cycles optimized for DNA synthesis.

To minimize depurination, each treatment with TCA should not be extended beyond the times specified in the cycles.

IMPORTANT Do not stop a synthesis while the DNA is exposed to TCA.

Some things to note:

Trityl On synthesis

If the synthesis is conducted Trityl On, for purification by OPC or trityl-on HPLC, the 5´ end fragment of an apurinic ammoniolysis product will bear a trityl group and may complicate purification.

Long oligos near3´ end

Long oligonucleotides which are purine-rich near the 3´ end8

may undergo depurination.


Coupling Chemistry Reactions

Washing and Dryingof Support

Before beginning the coupling step, the following steps are taken:

SimultaneousDelivery of

Phosphoramiditesand Tetrazole

According to the oligonucleotide sequence, one or more of the phosphoramidites (Bottles 1 to 8) and tetrazole are then simultaneously delivered to the column. When these reagents mix, the mild acid, tetrazole (pKa = 4.8), transfers a proton to the nitrogen of the diisopropyl group on the 3´-phosphorous (see figure below).

Step Action

1 The support is washed extensively with acetonitrile to make it anhydrous and free of nucleophiles (such as water).

Note Extraneous nucleophiles compete with the support-bound 5´ hydroxyls for the activated phosphoramidite and decrease coupling efficiency.

2 The support is then dried by an argon flush to remove residual acetonitrile.


This protonated amine makes a very good leaving group upon nucleophilic attack by the tetrazole to form a tetrazolyl phosphoramidite:9

Mixed sequence probes are synthesized by simultaneous delivery of more than one base (AGCT) and tetrazole with near-equivalent coupling:


Reactive intermediate

The tetrazolyl phosphoramidite is the reactive intermediate which forms the internucleotide phosphite with the support-bound 5´ hydroxyl.

Excess of tetrazole

An excess of tetrazole ensures that the phosphoramidite will be rapidly activated.

Excess of phosphoramidite

The excess of phosphoramidite relative to free 5´-hydroxyl ensures that the reaction is nearly quantitative (over 98% coupling).


Up to four bases Up to four bases may be specified as a mixed sequence probe. The four nucleoside phosphoramidites have slightly different reactivities.10

Reactivity order The cyanoethyl phosphoramidites follow the reactivity order of T > G > C > A.

Relative percentages

When all four are delivered simultaneously, their representation will be (normalized to 100%):T - 30%; G - 26%; C - 24%; A - 20%.

These values are slightly dependent on the following:

� Cycle

� Location of the site in the oligonucleotide

� Age of the phosphoramidite solutions, and other variables.

They have a range of about 3% because of these variables.


Capping Chemistry Reactions

Why Capping isNeeded

Because coupling is not always quantitative, a small percentage of support-bound nucleotides (usually less than 2%) can fail to elongate. Such support-bound nucleotides, however, are capable of propagating in subsequent coupling steps. As a result, these failure sequences would contain one less nucleotide than the full-length product and can be difficult to isolate.

Since the unreacted chains have a free 5´-OH, they can be terminated, or capped, by acetylation with acetic anhydride and 1-methylimidazole:11

Capping Process Although capping is not required for DNA synthesis, it is highly recommended because it minimizes the length of the impurities and thus facilitates product identification and purification (figure below).

Note The capping reagents, acetic anhydride, and 1-methylimidazole (NMI) terminate unreacted chains by acetylating the 5´-hydroxyl groups.


Non-failure sequences not affected

The chains which reacted with the phosphoramidite in the previous step are still blocked with the dimethoxytrityl group, so they are not affected by capping.

Failure sequence removed from synthesis

Capped failure sequences do not participate in the rest of the synthesis reactions.


As the figure on the previous page shows, capping proceeds as follows:

Note Initially, the two capping reagents need to be segregated since the active acetylating agent, acetylmethylimidazolide, is unstable. The capping time required to acetylate the 1 or 2% of unreacted 5´ hydroxyls is very brief, only a few seconds. It is important to minimize this time to prevent loss of cyanoethyl groups from the internucleotide linkages and to prevent base modification by-products. Studies at Applied Biosystems have demonstrated extensively the efficiency of both a shorter capping time and following the capping by oxidation.12

4

Stage Description

1 Equal volumes and equimolar amounts of two binary reagents, acetic anhydride and 1-methylimidazole (NMI), are delivered to the OneStep column where they mix to create a powerful acetylating agent.

2 This agent reacts at the 5´ hydroxyls rendering these moieties unreactive for the remainder of the synthesis.

3 The excess reagents are then removed by an argon flush (and ACN wash).


Oxidation Chemistry Reactions

Oxidation Reaction After coupling, the internucleotide linkage is oxidized from the phosphite to the more stable phosphotriester. The figure below shows the reaction that occurs when iodine—in tetrahydrofuran, water, and pyridine—oxidizes the trivalent phosphite to the stable pentavalent phosphate triester. This reaction is complete in less than 30 seconds.

Oxidation proceeds as follows:

IMPORTANT Do not stop the synthesis while the phosphorus is unoxidized.

Importance ofOxidation After

Capping

When oxidation occurs before capping, traces of water in the oxidizing solution can cause the formation of acetic acid from acetic anhydride during capping. The oligonucleotides are then exposed to acid and capping is less effective, as determined by scientists at Applied Biosystems.

Comparison of the enzymatic digestion/base composition assay on oligonucleotides made with different cycle orders—capping then oxidation and oxidation then capping—shows markedly different results. When capping immediately follows coupling11,13, a small side-reaction (the phosphitylation of the O-6 position of guanosine, is minimized.

The Applied Biosystems standard—capping then oxidation—gives far less, usually undetectable, amounts of base-modified nucleotides. Cycles with oxidation then capping give high levels of the mutagenic modified nucleotide, 2,6-diaminopurine.12

Stage Description

1 When the iodine-water-pyridine-THF mixture is delivered to the column, an iodine-pyridine complex forms an adduct with the trivalent phosphorus.

2 When this adduct comes in contact with water, it decomposes and a pentavalent phosphotriester internucleotide group results.


DNA Cleavage and Deprotection

Introduction When the synthesis is finished, the product and capped failure sequences are still attached to the support as phosphate-protected, base-protected phosphotriesters. The oligonucleotides must be cleaved from the support; complete deprotection is necessary to produce biologically active DNA.

After Synthesis, the following occur:

Cleavage Following synthesis, the DNA remains covalently attached to the support. The oligonucleotides are cleaved from the support by treatment with fresh, concentrated ammonium hydroxide.

As seen in the figure below, the cleavage occurs at the base-labile ester linkage between the linker of the support and the 3´ hydroxyl of the initial nucleotide. The cleaved DNA has a 3´ hydroxyl.

PhosphateDeprotection

The cyanoethyl protecting groups are removed by treatment with ammonium hydroxide. This occurs at the same time as cleavage, making phosphate deprotection very quick and simple (as shown in the figure).

Stage Action

1 The turntable rotates, moving the OneStep column from the synthesis head to the cleavage head.

2 Ammonia is then passed through the column, cleaving the DNA from the support.

3 After cleavage, the ammonia solution is sent to a coil heated to 65 °C for base deprotection.

4 Deprotection of base and phosphate occur.


Base Deprotection Base-protecting groups are removed when the DNA solution is heated to 65 °C for 1 hour in the heating coils. The deprotection process also cleaves the acetyl caps from the failure sequences.

Base deprotection is an ammonolysis reaction, in which ammonia acts as a nucleophile that attacks the carbonyl of the amide protecting groups. It is important to observe these guidelines in use of ammonium hydroxide:

Guideline Description

Fresh reagent For effective treatment, use fresh, concentrated ammonium hydroxide on the instrument.

Consistent‘ concentration

To ensure no decrease in ammonia concentration, store the reagent in a refrigerator, tightly capped.

When to discard ammonia

Discard the ammonia stock 30 days after opening.


References

1. Efcavitch, J.W. 1988. Automated System for the Optimized Chemical Synthesis ofOligodeoxyribonucleotides. In Macromolecular Sequencing and Synthesis,Selected Methods and Applications. Alan R. Liss, Inc. 221-234.

2. Beaucage, S.L. and Caruthers, M.H. 1981. Tetrahedron Letters 22: 1859–1862.

3. Matteucci, M.D. and Caruthers, M.H. 1981. Tetrahedron Letters 22:1859-1862.

4. Andrus, A. and McCollum, C. 1991. Tetrahedron Letters 32:4069-4072.

Aug. 29–Sept. 2, 1989. Solid Phase Synthesis, Oxford, U.K.

Jul. 29-Aug. 3, 1990. 9th International Roundtable Nucleotides. Nucleotides &Their Biological Applications, Uppsala, Sweden.

Applied Biosystems User Bulletin 61.

5. Sinha, N.D., Biernat, J., and Koster, H. 1983. Tetrahedron Letters 24:5843-5846.

6. Horn, T., and Urdea, M.S. 1988. Nucl. Acids Res. 16:11559-11571.

7. Tanaka, T. and Letsinger, R.L. 1982. Nucleic Acids Research 10:3249-3260.

8. Efcavitch, J.W. and Heiner, C. 1985. Nucleosides and Nucleotides 4:267.

9. Dahl, O. 1983. Phosphorus and Sulfur 18:201-204.

10. Zon, G., Gallo, K.A., Samson, C.J., Shao, K., Summers, M.F. and Byrd, R.A. 1985.Nucleic Acids Research 13:8181-8196.

11. Eadie, J.S. and Davidson, D.S. 1987. Nucleic Acids Research 15: 8333-8349.Farrance, I.K., Eadie, J.S., and Ivarie, R. 1989. Nucleic Acids Research17:1231-1245.

12. Applied Biosystems. 1988. The Effect of the Synthesis Cycle on the ChemicalAuthenticity of Synthetic DNA. Research News. 239702

13. Bauer, B.F. and Holmes, W.M., Nucleic Acids Research 17, 812. 1989.


Section 2 – Oligonucleotide Purification, Quantitation, and Storage

Topics Covered This section describes how oligonucleotides produced by the ABI™ 3948 System are purified and quantified, and provides guidelines for storage and infomation about alternative chemistries.


Topic See page

DNA Purification 3-20

Purification Stages 3-20

Characteristics of Purification 3-20

Other Purification Methods 3-20

Overview of Oligonucleotide Quantitation 3-22

UV Spectroscopy 3-22

ODU as a Measure of Concentration 3-22

Measurement of ODU and Concentration 3-23

Introduction 3-23

UV Hardware 3-23

Setting the Baseline Absorbance 3-24

Scaling Factor 3-24

Linear Range of Absorbance 3-24

Calculation of concentration (pmol/µL) by the ABI 3948 3-25

Handling of Extinction Coefficients in Different Software 3-25

Extinction Coefficients for Fluorescent Dye Labels 3-26

Storage of the Oligonucleotide 3-27

Storage for Later Use 3-27

Storage Guidelines 3-27

Alternative Chemistries 3-28

Other Monomers 3-28


DNA Purification

Purification Stages The ABI™ 3948 DNA synthesizer automatically performs a trityl-selective purification of crude DNA using the polystyrene OneStep column (which has an affinity for trityl oligonucleotides) as follows:

Characteristics ofPurification

The following are characteristics of purification:

Many PCR and sequencing reactions have been successfully run using DNA primers directly from the 20% ACN/water solution.

Other PurificationMethods

PAGE (polyacrylamide gel electrophoresis) and HPLC (high-performance liquid chromatography) can be used for purification but are subject to the following qualifications:

� PAGE and HPLC can provide a high level of purity, but require initial capital investment and are labor intensive and time consuming.

� A short oligonucleotide (<30 bases) made with typically high synthesis efficiency (>98% average trityl yield/cycle) may require less stringent purification. Efficient desalting and removal of non-nucleotide synthesis by-products may be sufficient purification.

Stage Description

Transfer After deprotection, the ammonia solution of the crude trityl oligonucleotide is transferred from the heating coils back through the synthesis/purification column, now located at the purification head.

Note Acetic Acid is added to the ammonia solution to neutralize it.

Retention of oligonucleotide

The trityl oligonucleotide product is retained.

By-products, failure sequences not bearing a trityl group, and other impurities are not retained and are passed out of the column.

Removal of trityl group

After water washing, the trityl group of the support-bound oligonucleotide is removed with a mild acid solution, 3% TFA/water.

Elution of purified oligonucleotide

The purified oligonucleotide is then eluted with about 1 mL of a 20% acetonitrile solution.


Stable to concentrated ammonia

The support material is stable to concentrated ammonia.

Ammonia provides a denaturing medium

The ammonia solution provides a denaturing medium, eliminating secondary structure, hydrogen-bonding, and coelution of partially complementary failure sequences.

Retention of trityl group

The trityl group is detached and retained in the column.

Desired sample eluted in small volume

The purified, fully deprotected oligonucleotide is eluted in a small volume of 20% acetonitrile in water, completely desalted and ready for use.


PAGE and HPLC are elaborated in detail in Evaluating and Isolating Synthetic Oligonucleotides, The Complete Guide, published by Applied Biosystems (Stock Number 239902).


Overview of Oligonucleotide Quantitation

UV Spectroscopy Nucleic acids of any variety are most easily quantified by UV spectroscopy, measuring at or near their UV absorbance maxima, about 260 nm:

ODU As a Measureof Concentration

A measurement of the absorbance gives a measure of the concentration of the solution, provided the molar extinction coefficient is known. The Optical Density Unit (ODU) is the unit of measure of the amount of oligonucleotide.

The following definitions apply to using ODU as the Unit of Measure:

:

Details Description

How measured A dilute aqueous solution of 1 mL or less, depending on the cuvette size, is measured by either scanning a region of about 200–350 nm or a single 260 nm wavelength measurement.

Characteristics of absorbance

A scan of an oligonucleotide will show broad absorbance with a maxima near 260 nm.

Definitions Description

ODU and Beer’s Law

ODU One ODU is the absorbance (typically measured at 260 nm) of a 1 mL solution of oligonucleotide in water or an appropriate buffer at a neutral pH range in a 1 cm pathlength cuvette.

Beer’s Law The measurement uses Beer’s Law to allow conversion of the absorbance reading to a molar amount. Beer’s Law states:

A = εCl, where A = Absorbanceε = molar extinction coefficientC = Concentration (mol/L)l = path length (cm) typically 1 cm

Molar Extinction Coefficients

Molar extinction coefficent

The exact molar extinction coefficient of a substance is a constant dependent on the UV absorbing properties of the chemical structure of that substance.

Major contributors to extinction coefficient

DNA contains four major contributors to the extinction coefficient; the four nucleobases A, G, C and T.

Each of these bases has a different extinction coefficient and a sample of synthetic DNA has (generally) a mixture of all four.

Criteria for ODU Measurement and Conversion to Mass or Concentration

Optimum measurement criteria

The ODU of an oligonucleotide is generally measured at the position when the absorbance is at a maximum (typically 260nm). Oligonucleotides that are very rich in either purines or pyrimidines may actually have absorbance maxima above or below 260 nm dependent on the composition.

Criteria for converting ODU to mass or concentration

Once the ODU reading is obtained, using the approximation that 1 ODU represents about 33 µg of single stranded DNA, the mass or concentration of an oligonucleotide can be determined provided the molecular weight of the oligo is known.


Measurement of ODU and Concentration

Introduction The ABI™ 3948 Nucleic Acid Synthesis and Purification System automatically measures the yield of purified oligonucleotide syntheses as follows:

Note The ABI™ 3948 System does not automatically determine the yield of a crude oligonucleotide synthesis as the concentration of the DNA is too high (8-12 ODU/mL) to determine an accurate value.

UV Hardware The ABI™ 3948 Nucleic Acid Synthesis and Purification System uses a 0.2 mL pathlength quartz flowcell to quantitate oligos. A low pressure mercury lamp emits light at a wavelength of 254 nm to measure the absorbance of the oligonucleotide in solution. This unit was chosen because of its compact size, reliability and ease of maintenance.

Stage Action

1 Purified oligos are eluted form the purification workstation in 20% aqueous acetonitrile and delivered to a 0.2 mm quartz flowcell within the UV station.

2 A lamp projects light at 254 nm through the flowcell and the absorbance at this wavelength is measured.

3 The ABI 3948 System Control software converts this measurement into ODU and picomole/mL values.

UVFlowcell

UVFlowcell

UV lamp powertoggle switch shouldbe in “ON” position


Lamp Lifetime The mercury lamp has an expected lifetime of one year and is replaced annually during preventive maintenance (PN ?). If the reference voltage (baseline UV reading) falls below 0.5 V, contact your local service engineer.

Setting the BaselineAbsorbance

The ABI 3948 System sets a baseline absorbance value for each measurement by filling the flowcell with water. During operation, the most recent baseline reading is subtracted from the oligo UV reading to give a corrected value:

ODU = (Baseline Water UV Reading – Oligo UV Reading) x 1.67

Scaling Factor The scaling factor 1.67 (or 5/3) is used because the hardware that reads the UV is scaled to 5 volt maximum reading but the UV circuitry is designed to output to a maximum of 3 volts.

As an example suppose the baseline water UV reading was 0.81 and the UV reading was –1.19. The difference between the two readings is 2.00, which when multiplied by 1.67 gives a final value of 3.0 ODU.

Oligos flow into the cell either as:

� A solution in 20% aqueous acetonitrile, if purified, or

� As a solution in ammonia, if crude.

Neither of these two solutions has appreciable absorbance at the measured wavelength.

UV DetectorLinearity

All UV detectors have a range of absorbance over which the response of the detector rises linearly with increasing absorbance of the solution:

For the ABI 3948 instrument, the linear range of the UV cell is 0.5 to 3 ODU. Above this range the response of the detector is non-linear and subject to increased error. Therefore crude (unpurified) oligos made on the ABI 3948 instrument should be re-quantitated for accurate measurement.


Range of ODUs Most measurements of ODU in laboratories are in the range 0.1 to 1.0 absorbance units.

Above or below optimum range

Above or below this range the measured value begins to deviate from the true value by an increasing amount


The graph below provides an estimate of how close the ABI™ 3948 System can come to approximate a bench top spectrometer. A random sample of oligos made on the ABI™ 3948 System were taken and re-quantified on a calibrated spectrometer.

The graph shows that over 90% of the large sample analyzed here lies in the range ± 10%. The exceptions to these are that the sample includes some crude oligos with ODU’s of >6.0. These have the largest error as they are outside the linear range of the detector.

Calculation ofConcentration

(pmol/µL) by theABI 3948 System

An ODU reading is converted to pmol/µL based on the specific sequence of the oligo made. The formula used for a sequence of length X is:

where ECbase X is the extinction coefficient of base X

For the ABI 3948 System the values for the extinction coefficient in this formula are shown as 1% of the actual extinction coefficient for the nucleotide at each position in the sequence as follows:

ECA = 154 ECG = 117 ECC = 73 ECT = 88

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

50

%

60

%

70

%

80

%

90

%

10

0%

11

0%

12

0%

Percent Difference from true value

Nu

mb

er

of

sa

mp

les

0

10000 x ODU(ECbase 1 + ECbase 2 +......+ ECbase X-1 + ECbase X)

pmol/µL =


Handling ofExtinction

Coefficients inDifferent Software

The instrument handles extinction coefficients differently in different software, as listed in the following table:

ExtinctionCoefficients for

Fluorescent DyeLabels

The relevant extinction coefficients for the available Fluorescent dye labels are

� 6-Fam - 18363

� Hex - 28207

� Tet - 14642

As an example, the 20 base sequence 5’ ATC TAC CTT GGC TGT CAT TG 3’ has three As, four Gs, five Cs and eight Ts. The sum of the extinction coefficients is therefore:

3 x ECA + 4 x ECG + 5 x ECC + ECT = 462 + 468 + 365 + 704 = 1999

If the reported ODU for this oligo was 3.1 then the pmol/µL would be:

Software Version Description

2.0 (NPR) Extinction coefficients of bottles 5–8 are taken as an average of the above four values i.e. )

These values are hard-wired into the image and are not editable.

2.20 (Post-NPR) Values for A, G, C and T are hard-wired into the image but bottles 5-8 are operator specified in the Instrument Preference Page to allow for different 5´labels having different extinction coefficients.

(154 + 117 + 73 + 88)

4= 108

31,0001999

= 15.5 pmol/µL


Storage of the Oligonucleotide

Storage for LaterUse

Most applications for synthetic oligonucleotides require less DNA than the typical 0.5 to 4 ODU of purified oligonucleotide that the ABI 3948 System produces. Fortunately, oligonucleotides can be stored easily, with little or no degradation for long periods of time.

Storage Guidelines Use the following guidelines for storage:

Note Except for oligonucleotides in dry storage, keep oligonucleotides cold to minimize degradation and bacterial growth.

When stored by the above means, oligonucleotides are stable for over a year. Avoid storing oligonucleotides in solutions that are mutagenic, oxidizing, or outside the pH range of 3–12.

Guideline Description

Storage refrigerated in solution

It is most convenient to store them refrigerated as a solution, in either a crude or purified state.

Storage frozen in 20% acetonitrile

Purified oligonucleotides may be stored frozen in the 20% acetonitrile solution.

Storage of crudes Oligonucleotides synthesized and collected crude may be stored in the concentrated ammonia solution.

Other liquid storage media

Other storage media include:

� Ethanol

� Acetonitrile

� Triethylammonium acetate (TEAA)

� Ethylenediamine tetraacetic acid (EDTA)

Dry storage Alternatively, oligonucleotides may be stored as a dried pellet in a clean, dry vessel such as a micro-centrifuge tube.


Alternative Chemistries

Other Monomers In addition to synthesis with the standard phosphoramidite monomers, other monomers may be used on the ABI 3948 System synthesizer. These other monomers include:

� Aminolink

� Biotin

� Deoxyinosine

� Phosphalink, and the

� Fluorescent amidites

Note For more information on the monomers and alternate chemistries listed above, visit the Applied Biosystems Nucleic Synthesis web page as described below.

Please note the following qualifications when using other monomers:

� When using Aminolink or Phosphalink, select the “Crude, DMT-Off” post synthesis option in the New Synthesis Order screen.

� When using the fluorescent amidites, use the “Syn-Pure x.xx Dyes” run protocol.

� When usinging Biotin, select the “Syn-Pure x.xx Biotin” run protocol.

Note The “x.xx” presented in the names above represents the version numbers of Dye and Biotin protocols on your instrument, which vary with date of purchase/software release.

Step Action

1 In your web browser, enter www.appliedbiosystems.com/techsupport as the URL and click "Open" or otherwise enable the web connection to the Applied Biosystems Products and Applications web page.

2 In the Products and Applications web page, click the "Nucleic Acid Synthesis" button.

3 In the Nucleic Acid Synthesis web page, connect with the web page for the chemistry reagent or other topic of interest by clicking the appropriate button.

Overview of Software Commands 4-1

Overview of Software Commands 4

In This Chapter

Topics Covered This section provides information on the command menus available from the Menu bar. A general knowledge of these commands is required to operate the ABI 3948 System Control application. In addition, the New Synthesis Order command contains information about the Synthesis Order form.

This chapter contains three sections:

Section See Page

File and Edit Menus 4-2

Synthesizer and Window Menus 4-20

RunFiles, Sample Labeling, Synthesis Order and Multi-Order Files 4-31

4

4-2 Overview of Software Commands

Section 1 – File and Edit Menus

Topics Covered This section describes the commands on the File and Edit menus and covers the following topics:

Topic See page

Overview of File Menu Commands 4-3

Menu Commands 4-3

Command-Key Commands 4-4

New Synthesis Order Command 4-5

Introduction 4-5

Types of Information 4-5

Description of Fields 4-5

Sequence Entry 4-6

Open Command 4-8

Introduction 4-8

Synthesizer Windows 4-8

Synthesis Orders 4-8

RunFiles 4-8

Multi-Order Files 4-9

Open Synthesizer Command 4-10

Dialog Box 4-10

Procedure 4-10

List of Synthesizer Window Views 4-11

Other File Menu Commands 4-13

Send to Synthesizer Command 4-13

Save a Copy In Command 4-13

Send Copy to Synthesizer Command 4-14

Save and Reopen Command 4-14

Import/Export Command 4-15

Export Procedure Icons 4-16

Generate Run File Command 4-16

Overview of Edit Menu Commands 4-17

Read Selection Command 4-17

Find Command 4-18

Find Same Command 4-18

Replace Command 4-18

Replace Same Command 4-19

Use Sounds Command 4-19


Overview of File Menu Commands

Menu Commands The File menu contains commands to perform the following tasks:

Command Description

New Synthesizer Order Creates a new Synthesis Order.

Open Opens a saved Synthesis Order or saved Synthesizer database file.

Opens a Run File for label printing.

Opens a text file to produce Multi-order files.

Open Synthesizer Establishes communication with a synthesizer.

Send to Synthesizer Sends all synthesis setup information from a Synthesizer window to a synthesizer.

Save a Copy In Saves a copy of all information from a Synthesizer to a Synthesizer database.

Send Copy toSynthesizer

Sends a database copy of synthesis setup information to a synthesizer.

Close Closes the selected open Synthesizer window, Synthesizer database, or Synthesis Order,

Close All Synth Orders Closes one or more open Synthesis Orders,

Save Saves changes made to a selected Synthesizer window, Synthesizer database, or Synthesis Order (Save).

CAUTION Be aware that using the Save command with a Synthesizer window to which you have made changes downloads those changes to the synthesizer. You may not want to do this. It is preferable to use the Save As command first, do your edits on the copy, and then use the Send Copy to Synthesizer command.

Using Save with a Synthesizer database or Synthesis Order saves the changes to a Macintosh file.

Save As Saves changes made to an active window to a new file, enabling the file to be renamed.

Save & Reopen Used to fill out multiple orders for the same customer.

Import Sequencea

a. This command reads Import Cycle for an Edit Cycle view or Import Procedure for an Edit Procedure view.

Used to import cycles/procedures/sequences created in another application to an open Synthesis Order or Synthesizer window.

Export Sequenceb

b. This command reads Export Cycle for an Edit Cycle view or Export Procedure for an Edit Procedure view.

Used to export cycles/procedures/sequences from an open Synthesis Order or Synthesizer window.

Generate Run File Generates a Run File for the synthesis.

Page Setup These are regular Macintosh® commands and are not described here.

If you need information on using standard Apple commands, see the Macintosh System Software User’s Guide that came with your computer

Print

Quit

File


Note If you have logged on to the synthesizer using a Monitor Access password, the only commands available are Open Synthesizer, Close, and Quit.

Command-KeyCommands

As an alternative for the mouse in choosing some commands, you can use the Command-key equivalent that appears next to some commands on the pop-up menu. For example, to choose the Open command, hold down the Command (�) key and press O.

Note If you have logged on to the synthesizer using a Monitor Access password, the only commands available are Open Synthesizer, Close, and Quit.


New Synthesis Order Command

Introduction This command opens an empty Synthesis Order like that shown below.

Types of Information The New Synthesis Order command is used to document synthesis order information so that it can be transferred to the synthesizer. Synthesis information includes:

� Sequence listing

� Names of cycles

� End procedure

� DMT state used for synthesis

Note The Synthesizer Order window (below) automatically displays information in the Order Date field.

Description of Fields The fields in the Synthesis Order are described in the table below.

These are the fields available from the File Menu:

Field Name Description

Customer Name A field to identify the customer.

Customer Address A field for the customer’s address.

Phone /Fax # A field for the customer’s phone and/or Fax numbers.

PO Reference A field for the customer’s purchase order number.

Accnt# The account number assigned to the customer by ABD.

Order Date The date the customer placed the New Synthesis Order. This date corresponds to the date shown on the computer’s General Controls in the Control Panel.

Comments Any information needed to further describe the sequence.

Note Entries in this field should be limited to 49 characters since only the first 49 characters are transferred to the RunFile or


Sequence Entry As you enter a sequence in the Sequence field, the number of A, G, C, T bases are counted in the bottom of the Synthesis Order form and the length of the sequence is listed in the left-hand column. The IUB ambiguity codes are counted as one base for the length of the oligonucleotide.

After you have entered a sequence, you can check your typed entry against a hard copy as follows.

When the Synthesis Order form is completed, you can save the order either as:

� A Synthesis Order (Save and Save As commands), or

� As a sequence file (Export Sequence command)

Synthesis Orders are used both as a record of a customer order and to input the sequence and other information needed for the instrument to produce the oligonucleotide.

Protocol Options Two pull-down menus are available on this line of the Synthesis Order form.

� The first menu allows you to assign any of 15 pre-defined protocols for production of the sequence.

� The second pull-down menu allows the following three choices for the oligonucleotide.

– Purify Oligo - When this choice (the default) is selected, the synthesized oligo is automatically purified.

– Crude - DMT Off - When this choice is selected, the oligonucleotide is not purified and the DMT group at the 5' end of the sequence is automatically removed.

– Crude - DMT On - When this choice is selected, the oligonucleotide is not purified and the DMT group at the 5' end of the sequence is left on.

Sequence Name This field allows you to enter up to 31 characters for the sequence name.

Sequence This field is where you enter the component oligonucleotides in the sequence. Besides the four bases, valid entries include 5, 6, 7, and 8 (bottle positions), and single-character IUB ambiguity codes.

These are the fields available from the File Menu: (continued)

Field Name Description

To check your sequence entry:

Step Action

1 Highlight the entry in the window.

2 Choose the Read Selection command from the Edit menu. A synthesized human voice reads the sequence back to you.


Note Be sure to end the sequence on the 3´ end with A, B, C, or T or you will be presented with the message shown in the figure below (synthesis starts at the 3´ end of the sequence and only A, C,G, or T are permissible entries).

When a Synthesis Order is opened in Microsoft Word or other word processors, it contains a complete record of the information entered into the sequence order. When a Synthesis Order is saved as a sequence file and then opened in Word (or other software), it contains only a listing of the sequence.


Open Command

Introduction This command opens a directory dialog box (figure below) that displays files created by the ABI 3948 System Control application and stored on the hard disk or on a floppy disk.

The types of files which may be opened by this command include the following:

� Synthesizer Window or database files

� Synthesis Order files

� RunFiles

� Multi-Order text files

SynthesizerWindows

A Synthesizer Window is the window used to control the ABI™ 3948 DNA Synthesis and Purification System. Complete information on the window is provided in Chapter 5 of this manual.

Synthesis Orders Synthesis Orders are the documents loaded into the Synthesizer Window to produce oligonucleotides. Information on this document is presented in several places in the ABI™ 3948 System User and Reference Manuals:

� Information about Synthesis Orders is provided under ““New Synthesis Order Command” on page 4-5 and under “About Synthesis Orders” on page 4-38.

� Information on preparing individual Synthesis Orders is provided under “Preparing Synthesis Orders” on page 4-8 of the ABI™ 3948 System User’s Manual.

� Information on the text files used to create multiple Synthesis Orders is contained in Appendix C of the ABI™ 3948 System User’s Manual.

RunFiles Information on RunFiles is provided in Section 3 of this chapter. Since RunFiles are used as a source for label information, Section 3 also provides the following information to support the process of label generation:

� Detailed information on the contents of RunFiles is provided under “RunFiles” on page 4-32.

� Information on generating labels using the ABI 3948 System Control application is provided under “Sample Labeling Feature” on page 4-33.

� Detailed information on the ABI 3948 System Control window used to generate labels is provided under “Label View Information” on page 4-36.


Multi-Order Files Information on Multi-Order files, which are text files used to produce multiple Synthesis Orders, is provided under“Multi-Order Files” on page 4-41 in this section. For detailed information on the syntax of such files, see Appendix C of the ABI™ 3948 System User’s Manual, “Creating and Using Multi-Order Files.”


Open Synthesizer Command

Dialog Box Establishes communication with an instrument on either a single zone or multi-zone network. When you choose this command, the Open Synthesizer Dialog box (below) opens.

This dialog box can display a multi-zone network, or list synthesizers on a single zone network.

Note The default application memory setting is 3048K. You can have a maximum of four databases and 48 orders open. If you need to open more windows, you will need to assign more memory to the ABI 3948 System Control application by selecting the application icon (application closed) and using Command-I.

Procedure To establish communication with a synthesizer:

Step Action

1 Choose Open Synthesizer in the File menu.

2 Select the zone name in the scroll box labeled Apple Talk Zones, if applicable.

3 Select a synthesizer in the box labeled Select a Synthesizer.

4 Click OK to present a password dialog box.

5 Enter a password and click OK.

A Synthesizer window like that shown on page 4-11 appears.


List of SynthesizerWindow Views

A pop-up menu at the top of the Communication View, the initial Synthesizer window, displays the views listed below the figure.

The Synthesizer window contains the following views which provide the options you need to define your synthesis run, monitor the run, manually control the run and edit cycles and procedures:

x.xxx

Name of View Purpose

Communication This is the initial view shown above.

Run Setup This view is used to set up a run.

Run Protocol This view is used to define the chemistry protocol to be used for a run.

Monitor Chemistry This view is used to monitor chemistry in progress during a run.

Monitor Instrument This view is used to monitor instrument hardware during a run.

Monitor Run This view is used to monitor the production of oligonucleotides during a run.

Edit Synthesis Cycle This view is used to create and edit synthesis cycles.

Edit Cleavage Cycle This view is used to create and edit cleavage and deprotection cycles.

Edit Purification Cycle This view is used to create and edit purification cycles.

Edit Begin Procedure This view is used to create and edit begin procedures.

Edit End Procedure This view is used to create and edit end procedures

Edit Bottle Procedure This view is used to create and edit bottle procedures.

Misc (Miscellaneous) Procedures This view is used to create and edit various types of procedures.

Manual Control This view is used to exercise manual control over the instrument.

Power Fail History This view reports power failures which occur on the instrument.


When you choose one of these views from the pop-up menu, the contents of the Synthesizer window change to display the related options.

Chapters 5 and 6 provide complete descriptions of all the views in the Synthesizer window and the available options.

Instrument Test This view is used to test instrument hardware.

B+Tet Calibration Table This view displays values used to provide maximum times for phosphoramidite deliveries.

Reagent Utilization Table This view is used to calculate the amounts of reagents required during run setup.

Name of View Purpose


Other File Menu Commands

Send to SynthesizerCommand

This command sends the contents of the Synthesizer window to a synthesizer. Use the command to send information to the ABI™ 3948 Nucleic Acid Synthesis and Purification System after creating new or modified versions of the following:

� Run Protocols

� Cycles - synthesis, cleavage/deprotection, or purification cycles.

� Begin, End, Bottle, or Shutdown procedures.

� Functions

� Time/Date setting

Note If you have made changes to a Synthesizer window and intend to save them, you should use the Save a Copy In command before using the Send to Synthesizer command, because this is the most direct way of saving your changes.

Save a Copy InCommand

This command makes a complete copy of the run setup, protocols and all other cycles, procedures, and settings for the ABI™ 3948 Nucleic Acid Synthesis and Purification System. It places the information in a computer file, which serves as a backup.

When you choose this command, an alert box like that above informs you that the copying process takes some time.

IMPORTANT The Save a Copy In command copies the contents of the Synthesizer database.

When you click OK to initiate saving a copy, a directory dialog box like that shown below appears.

Assign the file name and location in the directory dialog box.


Send Copy toSynthesizerCommand

This command sends a database file that contains all the information in an open database window to the ABI™ 3948 Nucleic Acid Synthesis and Purification System. The database file is created when you choose Save a Copy In... (see page 4-13).

This command is useful for:

� Transferring saved synthesizer information from one instrument to another instrument on the network.

� Restoring all information and instrument settings to a previous backup version. The combination of Save a Copy In and Send Copy to Synthesizer allows many users to share one synthesizer and still have completely individual customized setups.

When you choose this command, a dialog box (see below) displays the ABI™ 3948 instruments that are connected to the computer.

Note For multi-zone networks, the dialog box displays a zone list.

IMPORTANT You will need to recalibrate the sensors only when you reset the database. Sending a copy of a new database does not require a sensor calibration.

Save and ReopenCommand

This command is:

� Useful when filling out a number of Synthesis Orders for the same customer.

� Allows you to enter another sequence without reentering the other information on the form (as you would need to do if you used the New Synthesis Order form).

Note This command is like the Save and Save As commands except that the version of the Order remaining open does not contain any of the sequence information just saved.

Synthesizer-1


Import/ExportCommand

The Import and Export commands are available when you are creating a Synthesis Order or creating and editing cycles and procedures in a Synthesizer window. The command name changes to reflect the name of the capability that is currently active, as described in the table below:

Export ProcedureIcons

The Export Procedure command produces a specific type of file. Once exported, you can import a file type into any window that corresponds to the file type. The various file types and corresponding icons are shown in the figure below.

Command Where available

Import Sequence Synthesis Order

Import Cycle Edit Synthesis CycleEdit Cleavage CycleEdit Purification Cycle

Import Procedure Edit Begin ProcedureEdit End ProcedureEdit Bottle ProcedureMisc Procedure

Export Sequence Synthesis Order

Export Cycle Edit Synthesis CycleEdit Cleavage CycleEdit Purification Cycle

Export Procedure Edit Begin ProcedureEdit End ProcedureEdit Bottle ProcedureMisc Procedure

Sequence file or Synthesis Order icon

Cycle file icon

Begin Procedure file icon

End Procedure file icon

Bottle Procedure file icon

Miscellaneous Procedure file icon


Generate Run FileCommand

Although a RunFile (see below) is normally generated automatically at the end of a run, you are always prompted by the dialog box shown in the right side of the figure when you click the Load button in the Run Setup view.

The type of RunFile saved by clicking Yes in the dialog box is a TRunFile, the same type produced by the Generate Run File command (the keyboard shortcut for the command is Command - M).


Overview of Edit Menu Commands

Overview of EditCommands

The Edit menu commands perform the following tasks:

Besides choosing a command from a menu using the mouse, you can choose a command by using the Command key equivalent that appears on the menu next to the command. For example, choose the Read Selection command by holding down the � key and then pressing K.

Note If you have logged on to the synthesizer using a Monitor Access password, only the Open Synthesizer, Close, and Quit commands are available.

Read SelectionCommand

This command reads aloud the sequence entered into the New Synthesis Order. You can check the entered sequence by comparing the vocalized sequence to the original sequence.

Command Description

Undo Typing/Redo Typing Standard Apple Edit Menu Commands.

Note If you need information about these commands, see the Macintosh System Software Users Guide that came with the computer.

Cut

Copy

Paste

Clear

Read Selection Reads a sequence or portion of a sequence in the Edit Sequence window out loud (Read Selection).

Find/Find Same Find (or find again) a particular sequence of bases in the Edit Sequence view.

Replace/Replace Same Replace (or replace again) a particular sequence of bases in the Edit Sequence view with a designated sequence of bases

Select All Standard Apple Edit Menu Command (see Note above).

Use Sounds Turn on or turn off the sounds that accompany communication between the computer and the 3948 (Use Sounds).

Show Clipboard Standard Apple Edit Menu Command (see Note above).

To hear the sequence read aloud:

Step Action

1 Click the cursor on the sequence.

2 Use the Select All command to highlight the entire sequence, or drag the cursor across the sequence to highlight a portion of the sequence.

3 Choose Read Selection. A synthesized voice reads the order of bases entered in the Sequence field.

4 To interrupt the reading, press the Shift key. The reading stops and unread text remains selected.


Find Command This command locates a particular sequence of bases in an Synthesis Order.

Find SameCommand

This command locates the second and subsequent occurrences of the pattern specified by the Find command.

Replace Command This command finds one pattern of bases and replaces it with another.

To find a defined sequence:

Step Action

1 Click the cursor in the sequence.

2 Choose the Find command. A dialog box like that below appears.

3 Enter the pattern you want to find in the dialog box and click Find.

If the pattern is in the sequence, it is highlighted. If the pattern is not found, the computer emits a beep.

Note As a convenience, both Find and Replace ignore differences between lower and upper case letters. You may type everything in lower case. 3948 Control software automatically converts the text to upper case.

To replace one pattern of bases with another:

Step Action

1 Click the cursor in the sequence.

2 Choose the Replace command. A dialog box like that below appears.

3 Enter the pattern you want to remove in the field labeled Find what string?

4 Enter the sequence you want to substitute in the field labeled Replace with what string?

5 Click Replace to change only one occurrence of the base pattern.

Click Replace All to change all occurrences of the base pattern.

Click Find to locate each occurrence of the base pattern.


Replace SameCommand

This command repeats the substitution designated in the Replace dialog box (see figure above).

Use SoundsCommand

This command controls the built-in sounds that accompany communication on the network. The command acts like a toggle switch, alternating between the off and on positions.

The sounds verify that communication is occurring (and you may find them entertaining). However, the sounds use computer memory and can slow down communication. Communication is noticeably faster with the sound turned off.


Section 2 – Synthesizer and Window Menus

Topics Covered This section describes the commands on the Synthesizer menu, the contents and function of the Instrument Preferences window, and the purpose of the Window menu.


Topic See Page

Overview of Synthesizer Commands 4-21

Menu Commands 4-21

Abort Command 4-22

Procedure 4-22

Interrupt Command 4-22

Description 4-22

Resume Command 4-22

Description 4-22

Pause After Command 4-23

Introduction 4-23

Programming a Pause 4-23

Synchronize Clocks Command 4-24

Procedure 4-24

Change Password Command 4-25

Procedure 4-25

Change Name Command 4-26

Procedure 4-26

Instrument Preferences Command 4-27

Purpose of the Command 4-27

Auto-Resume Feature 4-27

Setup Variables 4-28

Setup Choices 4-29

Instrument Dip Switches 4-29

Window Menu 4-30

Description 4-30


Overview of Synthesizer Commands

Menu Commands The Synthesizer menu provides the commands listed in the table below:

Note These commands are not available if you have logged on to the synthesizer using a Monitor Access password.

Synthesizer Command Description

Abort This command terminates a run in progress.

CAUTION Aborted runs can not be resumed.

Interrupt This command interrupts a run in progress. Use this command when you intend to resume the run.

Note Relay 2 is turned on when a run is Interrupted, is turned off when the run is Resumed, and is pulsed for two-tenths of a second when a run ends. This is done to allow relay 2 to trigger an alarm or other device to alert the operator when a run has paused or ended.

Resume This command resumes an interrupted run.

Pause After This command is used to program a pause during a run. Runs halted with this command are started with the Start button, not the Resume command.

Synchronize Clocks This command is used to synchronize the instrument’s clock with the computer’s clock.

Change Passwords This command is used to change the passwords assigned to the instrument.

Change Name This command is used to change the name assigned to the instrument.

Instrument Preferences This command is important in setting up a run because it opens a special window used to control a number of instrument parameters.


Abort Command

Procedure This command stops synthesis immediately. After you use the Abort command, you must completely start over, setting up your run again using the Run Setup window.

The Abort command is used as follows:

Interrupt Command

Description The Interrupt command halts synthesis at the next Safe step and also disables Auto Resume. You can re-start synthesis after an interrupt with the Resume command.

Note A Manual Control action can not be interrupted but procedures can.

Relay 2 is turned on when a run is Interrupted, is turned off when the run is Resumed, and is pulsed for two-tenths of a second when a run ends. This is done to allow relay 2 to trigger an alarm or other device to alert the operator when a run has paused or ended.

Resume Command

Description This command re-starts a run after an interrupt.

Step Action

1 Choose Abort from the Synthesizer pop-up menu while a run is in progress. This will present the following dialog box:

IMPORTANT Be certain that you really want to abort the current synthesis since this command stops all active columns and you must completely start over again.

2 Click Yes if you want to abort the current run or click No if you have reconsidered.


Pause After Command

Introduction When you choose the Pause After command (Synthesizer menu) while a run is in progress, a dialog box like this is presented.

The Pause After command enables you to:

� Pause a run after any synthesis listed (and still not completed) in the dialog box.

In the figure example above, only one choice is possible for setting a pause after.

� Set a pause after a number of listed syntheses, if you use the command earlier in the run.

Programming aPause

A Pause After has the following characteristics:

When you are ready to save changes to your pause choices, do the following:

� Click the Save Pause Options button.

� After the system pauses at the programmed point, extend the current run as described in the User’s Manual in “Extending a Run” on page 2-35 of the ABI 3948 User’s Manual.


Execution after end of synthesis

A Pause After is executed only after the end of synthesis.

This is necessary because a turntable move is needed to load new columns and the synthesis jaws must remain sealed until synthesis chemistry is completed

Information in dialog box

Besides indicating where a run may be paused, the dialog box provides the following information:

� Syntheses completed (marked by *1)

� Synthesis currently in progress (marked by *4)

� Whether a synthesis is scheduled (marked by *2) or not scheduled (marked by *3).

Set All button The Set All button allows you to set a pause after every available position

Clear All button The Clear All button clears any choices you have made.


Synchronize Clocks Command

Procedure This command resets the clock on the ABI™ 3948 instrument to match the clock on the computer. When you choose the command, a dialog box like that below appears.

T

To synchronize the synthesizer and computer clocks:

Step Action

1 Choose the Synchronize Clocks command in the Synthesizer menu.

2 Check the date and time that appears after the word Macintosh.

If it is not correct, click Cancel to close the dialog box. Reset the current date and time on the computer’s Control Panel. See the Macintosh System Software User’s Guide for instructions.

3 When the Macintosh time and date are correct, click OK in the dialog box. The synthesizer’s time and date settings change to match the Macintosh settings.

8

8


Change Password Command

Procedure This command is used to assign or change a password.

To assign or change a password:

Step Action

1 Choose the Change Password command from the Synthesizer menu.

This brings up the dialog box to the left below.

2 Do one of the following:

� If no password has been assigned yet, click OK without entering a password.

� Enter the Full Access password if one is assigned.

Either of these actions presents the dialog box shown below:

Note If you enter the wrong password when one has previously been assigned, the dialog box shown to the right in step 1 appears.

3 Do one of the following:

� For Full Access, click the Full Access radio button and then click the Continue button.

� For Monitor Access, click the Monitor Access radio button and then click the Continue button.

Either of these actions will present the dialog box shown to the left below.

4 Re-enter the password you entered in step 2. If you fail to enter the same password, you will be presented with the dialog box shown to the right above.


Change Name Command

Procedure This command assigns a name to a synthesizer. The Synthesizer name appears when you choose the Open Synthesizer command to establish communication with the synthesizer, and in the title bar of the Synthesizer window.

When you choose the Change Name command, a dialog box appears like that shown below.

Do the following to change a name:

Step Action

1 Type a synthesizer name in the entry field labeled Name. The entry field holds up to 32 characters.

2 Click OK to finish assigning a name.


Instrument Preferences Command

Purpose of theCommand

The command is used to present the Instrument Preferences window, shown below. This window allows a number of chemistry and hardware settings to be made in one place:

The ability to make a number of changes in one place facilitates adjusting the instrument to the specific environmental conditions found in different labs. The Instrument Preferences window also is intended to support the Auto-Resume instrument feature.

Auto-ResumeFeature

The Auto-resume feature, described under “Auto-Resuming” on page 2-17 of the ABI 3948 System User’s Manual, is intended to strike a balance between throughput and reliable operation. See “Auto-Resuming” in the referenced material for more information on using the Auto-resume feature.

Type of Setting Description

Setup Variables

Setup Variables allow the user to change values for key functions in chemistry cycles.

Setup Choices Setup Choices allow a number of choices for hardware or operation conditions.

Instrument Dip Switches

Instrument Dip Switches show the settings of dip switches on the processor (CPU) board and should only be changed by an ABD Service Engineer.

656033380

15200

130

35


Setup Variables Entries made for Setup Variables directly change parameter values of a number of key functions in chemistry cycles. This capability allows the user to configure the instrument for different operational conditions. The values shown in the Instrument Preferences example on page 4-27 are the default values and can be changed by selecting the existing entry and typing in a new value.

Note Values entered for Setup Variables in this window are default values: entering a “non-zero” value into the Misc field of the corresponding step of the associated cycle supersedes the Instrument Preferences setting.

Functions affected include:

Function or Parameter Description

Deprotect Temp (170) This variable (for Function 170, “Start Depro Htr”) sets the final temperature at which the deprotection coils are set for deprotection. Final temperature is set here rather than by the associated step in the Cleavage cycle.

Deprotect Mins (169) This variable (for Function 169, “Depro Htr Wait”) sets the minimum time that the deprotection coils will remain hot once the final deprotection temperature has been reached. This time is set here rather than by the associated step in the purification cycle.

Xfer Into Coil (260) This variable (for Function 260, “Set Coil Temp”) sets the temperature at which the deprotection coils are set to for the transfer of the sample from the cleavage vessels into the Deprotection coils. This temperature is set here rather than by the associated step in the purification cycle.

Xfer From Coil (261)/ Coil Cool Secs (261)

These variables (for Function 261, “Wait Coil Temp”) sets the target temperature for the deprotection coil and the time allowed for the temperature to be reached before the transfer from the coils to the purification columns. Temperature and time are set here rather than by the associated step in the purification cycle.

Jaw Leak Test in PSI This variable sets the pressure used for the Jaw/block leak test that is executed when the jaws close after each turntable move at the beginning of each cycle.

Leak OK 0.01 PSI This is the maximum passing leak rate for the Jaw/Block Leak Test in 1/100 of PSI.

Auto-resume Minutes This is the time in minutes that the system waits before auto-resuming from a failed sensor delivery.

Auto-res OK 0.01 PSI This is the maximum jaw leak in 1/100 PSI allowed to keep auto-resume enabled - may not be as large as the Leak OK value.

Ext. Coefficient 5

Ext. Coefficient 6

Ext. Coefficient 7

Ext. Coefficient 8

The values for these parameters represent 1% of the extinction coefficient values for the contents of Bottles 5-8. These values are used to convert ODU values to pmole/mL.

These values can be adjusted to give more accurate quantitation when using bottles 5–8 (default = 10800).


Setup Choices These checkboxes provide a central place to make the following hardware or operational settings.

Instrument DipSwitches

These are hand Dip switches on the CPU board whose settings are reflected on this page. They may only be set by taking off a side panel and flipping the switches on the board.

Setup Choice Description

User 4x12 Tube Rack Check this box when you want to use the white rack (4X12 configuration). Leave the box blank to use the red rack (8X6 configuration). This box is unchecked by default.

Pause On Sensor Fail The instrument will pause on a failed sensor delivery with this box checked. The box is checked by default. The system may auto-resume from this pause if auto-resume is enabled.

Pause On Jaw Leak The instrument will pause on a failed leak test with the box checked (unchecked by default). The system will not auto-resume from this pause.

End Row on Jaw Leak The affected turntable row will drop out of the run on a failed jaw leak test but the run will continue processing other rows. This box is checked by default.

Man Cont Jaw Testing This parameter enables jaw/block testing in Manual Control mode. This box is unchecked by default.

Log Dry Sensor Fxns This parameter enables “dry” sensor flows (flushes and backflushes) report to the Microphone log - normally only “wet” sensor deliveries report. Default is unchecked.

Note Be aware that enabling this parameter will produce a much larger Microphone file.

Mnfg. Jaw Test Mode Only used during instrument manufacturing.

Dip Switch Description

Check Illegal Values This parameter is enabled by default. It makes sure no “illegal” value combinations are being attempted (especially by User functions).

No Flow To Open Jaws This box is checked by default since this function is only used to test deliveries to the jaws.

Reserved Reserved for future use.


Window Menu

Description The Window menu displays all open Synthesis Orders, Database, Synthesizer, and Print Label windows. When you can select one of the items listed, that window moves to the front of the monitor display and becomes the active window.

Note A Print Label window is a window opened by opening a RunFile window in the ABI™ 3948 System Control application. Information presented for such files includes date of creation (in month, day, year) and time of creation (hour, minute, second, AM or PM). For more information on RunFiles, see “RunFiles” on page 4-32.

Window


Section 3 – RunFiles, Sample Labeling, Synthesis Order and Multi-Order Files

Topics Covered This section describes RunFiles, how to use RunFiles to label samples, and Synthesis and Multi-Order files.


Topic See Page

RunFiles 4-32

Information in the RunFile 4-32

Sample Labeling Feature 4-33

Procedure 4-33

Label View Information 4-36

Introduction 4-36

Information in the Five Columns 4-36

Information in Individual labels 4-36

Label Preferences Button 4-37

About Synthesis Orders 4-38

Introduction 4-38

Ways of Creating Synthesis Orders 4-38

Synthesis Order Sequence Entry 4-39

Importing and Exporting Sequences 4-39

Actions after Sequence Entry 4-40

Multi-Order Files 4-41

Introduction 4-41

Applications Used to Create 4-41

Generating Synthesis Orders 4-43


RunFiles

Information in theRunFile

The ABI 3948 System Control application generates a RunFile for each run. Run information is normally automatically generated and stored in a file like that shown below. The information on which a RunFile is based is no longer available from the instrument after the start of the next run.

The following types of information are provided in the RunFile log for each oligonucleotide produced:

� Column position, sequence name and listing for the sequence in each loaded position

� Sample Collector position for labeling the samples

� Begin and End procedure names

� Two measures of the quantity of each oligonucleotide; ODUs (Optical Density Units) and concentration in picomole/µL

� Name of the Synthesis cycle used

� Name of the Cleavage cycle used

� Name of the Purification cycle used

� Date of completion of Synthesis order (date Synthesis order was generated)

Note You can open a RunFile in two ways, by using Simple Text or by using the control application. When opened in Simple Text by double-clicking the File icon (Simple Text must be present on the Macintosh), the RunFile looks like the example in the figure below. Opening the RunFile with the ABI 3948 System Control application allows you to print labels.


Sample Labeling Feature

Procedure The ABI 3948 System Control application now has the capability of printing labels for your sample vials. This feature uses the RunFile created by the instrument as the source of the information needed for labeling.

Note If you can’t find a Run file after a run, use the “Generate Run File” command (page 4-16) to generate a new Run file.

To print labels for a run:

Step Action

1 Open the Run file generated by the sample run using the Open command.

A Oligo Labels dialog box like that shown below appears. For an explanation of the information in this view and other views shown in this procedure, see “Label View Information” on page 4-36.

2 To select a different dilution volume, click the Dilution Volumes button to present the Dilute Volumes view:

Note The dialog box shown in the figures in steps 1 and 2 has two views, the Oligo Labels view shown above, and the Dilute Volumes view shown below.


3 Scroll the Final Concentration scroll bar (located in the top middle of the Dilute Volumes view) to select the concentration.

You can choose any value in the range of 0.0 to 100 pmol/pmol/µL in 0.5 pmol steps. As you scroll, notice that the amount of reagent needed to dilute each sample changes in the sample listing below.

4 Once the dilution volume is selected, return to the Oligo Labels view by clicking the Oligo Labels button and then scroll to the bottom of the window.

After scrolling, the Oligo Labels view looks like the figure below. This portion of the Oligo Labels view is the Label print area which lists the information to be printed on labels.

5 To see the details of any label in the Label print area, select it to present the contents as shown in the figure above.

Note The contents of the label selected to the upper left are shown in an expanded form at the top of the window. See “Information in Individual Labels” on page 4-36 for a description of label contents.

6 Click the Label Preferences button to present the dialog box shown below.

Note See “Label Preferences Button” on page 4-37 for more information about the contents of this dialog box.

To print labels for a run: (continued)

Step Action


Note An alternate way of opening the Oligo Labels dialog box is to drop a RunFile icon onto the icon for the application. This opens the controller application and inputs the selected RunFile.

7 If desired, change the information displayed on each label as follows:

� Change the type of information displayed on the fourth line by clicking the radio button for a new type of information.

� Change the Misc Display Options to enable or disable the display of concentration information:

– Checking the Show pmol values checkbox enables the display of this type of information.

– Checking the “Show odu values” checkbox enables the display of this type of information.

8 Print out a single sheet of labels at the default X- and Y-axis settings to determine if label printing is correctly positioned.

9 Print another sheet of labels to determine if labels print properly:

� If label information is clearly centered and visible, you are done.

� If label information does not print properly, make another setting in step 8 and print out another sheet of labels to check printing.

To print labels for a run: (continued)

Step Action

If the label information... Then...

is clearly centered and visible you are done.

is too far to the left use the upper Printer offset scroll bar to enter a positive X-axis value.

is too far to the right use the upper Printer offset scroll bar to enter a negative X-axis value.

is too far up use the lower Printer offset scroll bar to enter a negative Y-axis value.

is too far down use the lower Printer offset scroll bar to enter a positive Y-axis value.

is not oriented properly check the orientation using the Page Setup command (File menu) and reset it to “Portrait” if necessary.

is too small or too large check the Scale value with the Page Setup command and set it to exactly 100%.


Label View Information

Introduction This section provides the following information on the label print views used in the procedure above:

� Information in the Five Columns

� Information in the Individual Labels

� Label Preferences Button.

Information in theFive Columns

The information shown in the five columns of the listing in the Labeling views in steps 1 and 2 above is as follows:

� Column 1 - Sample number

� Column 2 - rack position

� Column 3 - Sample name

� Column 4 - Crude/Purified (when field is blank)

� Column 5 - Dilution volume in µL

Information inIndividual Labels

Notice that the information contained in a selected label is presented at the top of the window. To see the contents of any label in the body of the window, select the label in the portion of the Oligo Labels view shown in step 4.

Note The example in the procedure shows the label contents corresponding to rack position A1 at the top of the window.

These types of information are shown in each label:

� Chemistry protocol name on the top line.

� Rack position (A1, etc., at the start of line two.

� Concentration in pM/µL (picomole/microliter) at the end of line two.

� Date of run at start of line three (011496 = November 4, 1996).

� Concentration in ODUs at the end of line three.

� Line 4 is blank until entry is made using the Label Preference button.


Label PreferencesButton

The Label Preferences button presents the Label Preferences dialog box, shown in step 6, which enables you to do the following:

Task Description

Centering label information

Adjust the x- and y-axis offsets for the printer so that label information can be centered on the label.

Specifying contentsof fourth line

Specify the content of the fourth line of the label and adjust the position of printing so that label annotation is correctly centered on the label

Specifying type of information on fourth line

Specify the type of Information presented on the fourth line of each label, which includes:

� Customer name

� PO (Purchase Order) number

� Account number

� A brief comment (it must fit on the fourth line)

Specifying additionalinformation

Specify that pM/µL and ODU information be presented on labels by checking the appropriate checkbox.


About Synthesis Orders

Introduction The Synthesis Order is the document used to input sequences into the ABI™ 3948 Synthesis and Purification system. A Synthesis Order documents other details about the run used to produce it such as:

� Customer name

� Address

� Phone or Fax number

� Order date

� PO number

There are three Synthesis Order formats:

� The single order file format

� The short multiple order file format

� The long multiple order file format

The single order file is the type of Synthesis Order created in the Control application by the New Synthesis Order command from the File menu. The two multiple order formats are alternative ways to generate single order files. Text files using the short or long multiple order file formats are called Multi-order files.

Ways of CreatingSynthesis Orders

The most direct way of creating a Synthesis Order is to use the New Synthesis Order command from the File menu of the ABI 3948 System Control application. This creates orders one at a time with a separate Synthesis Order required for each oligonucleotide synthesized.

An example of a Synthesis Order is shown below. The Synthesis Order is described in detail under “Types of Information” on page 4-5.

The Multi-order file capability was added to the ABI 3948 instrument application to expedite creation of multiple Synthesis Orders as follows:

� Creating Multi-order files is a two-step process which requires you to create a text file in the proper format, either the short or long format, and then open the files in the ABI 3948 instrument application.


� After the ABI 3948 System application opens a Multi-order file, the application is used to generate “single” Synthesis Order files for input to the instrument. An example of an opened Multi-order long format file is shown below.

Note For a detailed description of Multi-order file formats and how to use the Multiple Order window, see Appendix C of the ABI™ System 3948 User’s Manual, “Creating and Using Multi-Order Files.” Synthesis Orders, however created, appear like the example shown in the figure on “Ways of Creating Synthesis Orders” on page 4-38.

Once single format Synthesis Orders are created, they can be sent to the Synthesis database during run setup and then downloaded to the instrument when the run starts. For more information on this, see the instructions provided in Chapter 2 of the ABI™ 3948 System User’s Manual (“Assigning Sequences for the Run” on page 2-22).

Synthesis OrderSequence Entry

When you directly create a single order file using the New Synthesis Order command, a sequence can be input into the order in one of three ways:

� By typing it in

� By importing a sequence from a file using the Import Sequence command, or

� By using cut and paste to transfer the sequence from other applications

Once you have input a sequence, you can use it as is or you can edit it.

Note One type of edit is to change the A, G, C, and T letter representation to bottle position representation (5, 6, 7, 8).

Besides entry of the sequence in terms of the four bases, sequences can be entered or imported using mixed bases as a designation for a base site or ambiguity characters can be used to designate a base site.

Importing andExportingSequences

Upon opening a new Synthesis Order, the Import Sequence command (File menu) is available to import sequences. This command, however, can only be used to import sequences previously exported to a file to be archived from the ABI 3948 System Control application.

Once you have completed a Synthesis Order, the Export Sequence command is available to write a plain text file containing only the sequence for use as input to all programs that accept plain text input.

IMPORTANT The Export Sequence command is probably not the best way of maintaining a library of sequences since it provides just the sequence text.


Rather than exporting a sequence, it is better to use a Synthesis Order file to store each sequence since you are provided with fields for:

� Customer information

– Name

– Address

– Phone and Fax number

� Purchase Order and Account information

� Order Date

� Comments

� Protocol Option

� Sequence name

� Numbers of each of the bases or bottle positions 5 through 8

Synthesis Orders can be opened as well in many other applications.

Actions afterSequence Entry

Once you have created or edited a sequence, you can:

� Highlight the sequence listing and read it back using the Read Selection command (Edit menu) to ensure that it was entered correctly.

� Save the Synthesis Order containing the sequence for use in producing an oligonucleotide in the instrument

� Print out a copy, or

� Export the sequence to a file to be archived


Multi-Order Files

Introduction Multi-order (multiple Synthesis Order) files are discussed in this chapter because they may be opened by the Open command. Only general information is provided here. For a detailed description of the use of Multi-order files, see Appendix C of the ABI™ 3948 User’s Manual, “Creating and Using Multi-Order Files.?

Multi-order text files are created in either of two formats by a text processor and have the following characteristics:

Types ofApplications Used to

Create

All Multiple Order files may be generated any of the following types of applications:

� A text editor or a word processor that has its formatting capabilities disabled

� A stand-alone application such as a Web page order entry program

� A spreadsheet or database management program, especially one with programming or scripting capabilities

Multiple Synthesis Order files appear as shown below when opened with the ABI 3948 System Control application Open command:


Correct syntaxand starting tag

Multi-order files must have the correct syntax and start with a SYNTHMOSFORMAT tag for the short format or a SYNTHMOLFORMAT tag for the long format.

Limitation of short format

The short format only provides the capability to name the resulting oligonucleotide and list the sequence.

Additional long format capabilities

The long format additionally provides the capability to:

� List the names of the customer and synthesizer.

� Add comments.

� Set the DMT and PUR options.

Maximum number of Synthesis Orders

The maximum number of Synthesis Orders which can be created with a single Multiple Order file is 999.


Note Both Long and Short Format files appear as shown in the figure but the Long Format file will create Synthesis Orders with more content when additional entries are made in the text file.

GeneratingSynthesis Orders

Once a Multiple Order file is created, in either format, all that is required to generate single order Synthesis files is to open the file from the ABI 3948 System Controller application and then click the Make Order Files button (see figure on previous page).

Communication View and Operational Views 5-1

Communication View and Operational Views 5

In This Chapter

Topics Covered This section provides detailed information on a number of Synthesizer views used to operate the instrument from the ABI™ 3948 System Control application. The pop-up menu in all views is used to access any other Synthesizer view.


Topic See page

General Information about Operational Synthesizer Views 5-2

Using the Run SetUp View 5-20

5

5-2 Communication View and Operational Views

Section 1 - General Information about Operational Synthesizer Views

Contents of Section 1 Section 1 provides referrence information on the operational Syntheisizer Views and covers the following topics:

Topic See page

About the Synthesizer Window 5-4

Introduction 5-4

Access to Views 5-4

Type of Information in This Chapter 5-5

Keyboard Shortcuts 5-5

Communication View 5-6

Initial Database Window 5-6

Accessing the Synthesizer Window by Password 5-6

Run Setup View 5-7

Tasks Performed 5-7

Accessing the Run Setup View 5-7

Menus and Buttons 5-8

Run Protocol View 5-9

Used to Create Protocols 5-9

Protocol Name 5-9

Synthesis Cycle 5-9

Cleavage Cycle 5-9

Purification Cycle 5-9

Monitor Chemistry View 5-9

Introduction 5-9

Three Chemistry Panes 5-12

Description of Sequence Pane Information 5-13

Status and System Messages 5-13

Using Monitor Chemistry Information 5-14

Monitor Instrument 5-15

Introduction 5-15

Current Date and Time 5-15

Valves 5-15

Liquid Sensors 5-16

Conventions 5-16

Synthesis Jaw/Valve Block/Manifold Sensors 5-16

Cleavage Jaw Sensors 5-17

Deprotection Coil Sensors 5-17

Purification Jaw Sensors 5-17

Phosphoramidite Sensors 5-17

UV Detector Sensor 5-17

Pressure Regulators 5-17


Other Parameters and Instrument Conditions 5-18

Monitor Run View 5-19

Introduction 5-19

Types of Information Provided 5-19

Topic See page


About the Synthesizer Window

Introduction The Synthesizer window of the ABI 3948 System Control application:

� Appears on the computer monitor whenever you establish communication between the computer and a ABI 3948 System.

Note Up to four instruments at a time may be used with a single Macintosh.

� Contains all the tools you need to control and monitor the ABI™ 3948 Nucleic Acid Synthesis and Purification System.

The initial Synthesizer window, shown below, is the Communication view. This view appears whenever the ABI 3948 System Control application accesses an instrument.

Access to Views A pop-up menu at the top of the Synthesizer window displays the following views:

2.20x.xx

Pop-up menu

List of Synthesizer Window views:

View Purpose

Communication Initial view listing software version

Run Setup Used to set up a run

Run Protocol Used to create new protocols

Monitor Chemistry Used to monitor chemistry during a run

Monitor Instrument Used to monitor instrument hardware during a run

Monitor Run Used to monitor the Synthesis Orders produced during a run

Edit Synthesis Cycle Used to create and edit Synthesis cycles

Edit Cleavage Cycle Used to create and edit Cleavage and Deprotection cycles

Edit Purification Cycle Used to create and edit Purification cycles

Edit Begin Procedure Used to create and edit Begin procedures

Edit End Procedure Used to create and edit End Procedures

Edit Bottle Procedure Used to create and edit Bottle Procedures

Misc (miscellaneous) Procedure

Used to create and edit other types of procedures

Manual Control Used to manually execute functions


Type of Informationin This Chapter

To avoid presenting information in the same way in both the User and Reference manuals, Chapter 2 of the ABI™ User manual provides procedural information for the Synthesizer window and this chapter of the ABI™ Reference manual may provide procedures but primarily provides user interface details.

Keyboard Shortcuts As an alternative to the pop-up menu in the Synthesizer Window, you can simultaneously press the Command key (�) and one other key on the computer keyboard to see nine of the views.

Power Fail History Provides a listing of instrument power failures

Instrument Test Used to test instrument hardware

B+Tet Calibration Table Lists maximum times for B+Tet deliveries

Reagent Utilization Table Lists volumes for various reagent deliveries

List of Synthesizer Window views: (continued)

View Purpose

Synthesizer Window View Keyboard Shortcut

Run Setup c 1

Run Protocol c 2

Monitor Chemistry c 3

Monitor Instrument c 4

Edit Synthesis Cycle c 5

Edit Cleavage Cycle c 6

Edit Purification Cycle c 7

Edit Bottle Procedure c 8

Manual Control c 9


Communication View

Initial DatabaseWindow

The initial instrument database window which appears is the Communication view.

The Communication view of the Synthesizer window appears when you choose an instrument and a password dialog box is presented as the window appears.

Two types of passwords provide access to the Synthesizer window:

� Full Access - provides access to all views of the window

� Monitor Access - provides access only to the Monitor Chemistry, Monitor Instrument, Monitor Run, and Power Fail History Views.

Accessing theSynthesizer Window

by Password

Open up a Synthesizer window as follows:

2.20x.xx

Step Action

1 Choose the Open Synthesizer command and select the instrument to be opened from the dialog box. This will present the Communication view and a Password dialog box like that shown to the left below:

2 Type in the password in the dialog box and click OK to open access to the Synthesizer database:

� If no password has yet been assigned, click OK without entering a password.

� If an incorrect password is entered, the dialog box shown to the right below replaces the first dialog box. Clicking OK in this dialog box presents the dialog b ox to the left again.

Note You must enter the correct password to get past this point.


Run Setup View

Tasks Performed This subsection provides general interface information on the first page on the Run Setup View. For information on other Run Setup view windows, see Section 2 of this chapter.

The Run Setup view, shown below, enables you to perform the following tasks:

Accessing the RunSetup View

The Run Setup view is chosen either by executing this command from the Communications view menu or by the Command - 1 key sequence.

Task Description

Select sequencesfor the next run

Select/remove sequences to be used for the next run.

Assign chemistry and sort sequences

Assign chemistry protocols and sort the sequences by protocol to determine turntable loading positions.

Determine if sufficient reagents are on instrument

Determine if additional reagents are needed for the next run.

When insufficient reagents are available for the entire run, the options are:

� To replace reagents before the run

� To program a Pause After to replace reagents

Install OneStep columns

Place OneStep columns in the proper positions on the turntable to prepare for the run.

Prepare for run extension

Program a “Pause After” during a run to occur after synthesis has completed on a designated turntable row in preparation for run extension

Perform setup needed to extend a run

Enter sequences and load columns needed for run extension during a programmed system pause and then restart the run.


Menus and Buttons Various details of the Run Setup view are described in the following table:

Run Setup View Pop-Up Menus and Buttons

Pop-up Menus and Buttons Descriptions

Open This button is used to input Synthesis Orders into the Sequence Order list.

Del From List After you have moved sequences into the Sequence Order list using the Open button, the Del From List button is used to remove any selected sequence from this list.

Add+ Click the Add + button to transfer one or more selected sequences from the Sequence Order List to the Run Setup list.

Remove Click the Remove button to transfer a selected sequence from the Run Setup Order box back to the Sequence Order List.

Protocol Pop-up menu This pop-up menu allows a protocol to be assigned to a selected row in the Run Setup list.

AutoSort This button is used to autosort the sequences entered into the Run Setup list to maximize throughput on the instrument. See "AutoSort Procedure" on page 2-24 of the ABI™ 3948 System User’s manual for more information

Begin and End Procedure Pop-up Menus

These menus allow a different Begin and End procedure to be assigned in this view, but only if you have previously developed them.

Bottle Usage This button presents the Bottle Usage window to determine if sufficient reagents are present on the instrument to accommodate the current run. See "Checking Reagents and Required OneStep Columns" on page 2-27 of the ABI™ 3948 System User’s manual for more information.

The Bottle Usage window also indicates how many of each type of OneStep column is required.

Load This button presents the Load window enabling OneStep columns to be properly placed on the instrument. See "Loading" on page 2-30 of the ABI™ 3948 System User’s manual for more information.

Extend Run This button is used to extend a run when the run has been interrupted by a programmed “Pause After.” See "Using “Pause After” During a Run" on page 2-35 of the ABI™ 3948 System User’s manual for more information.


Run Protocol View

Used to CreateProtocols

The Run Protocol view is used to create protocols from the available cycles and procedures. This view initially looks like the figure below:

Each protocol comprises: a Synthesis Cycle, a Cleavage Cycle, and a Purification Cycle. Use the Run Protocol view as follows:

� When you select a protocol from the Protocol Name pop-up menu, its component cycles and procedures appear.

� If you select an undefined protocol, its component cycles and procedures become the default values listed for the standard protocol.

� To create a new protocol, enter a new name for the protocol, then choose the cycles and procedures from each pop-up menu, and click the Change button.

Note The Edit Synthesis Cycle, Edit Cleavage Cycle, and Edit Purification Cycle views are available to develop new cycle chemistry.

Protocol Name This pop-up menu lists the standard protocols (standard, dye-labeled, and biotin) and the additional undefined protocols.

Synthesis Cycle This pop-up menu lists standard Synthesis Cycles and additional undefined cycles. Use the Edit Synthesis Cycle view to define a new Synthesis Cycle or edit an existing one (see page 6-17).

Cleavage Cycle This pop-up menu lists standard cycles and additional undefined cycles. Use the Edit Cleavage Cycle view to define a new Cleavage Cycle or edit an existing one (see page 6-18).


Purification Cycle This pop-up menu lists standard cycles and additional undefined cycles. Use the Edit Purification Cycle view to define a new Purification Cycle or edit an existing one (see page 6-19).


Monitor Chemistry View

Introduction The Monitor Chemistry view, shown below, has the following four main areas:

Note This is the only view that will not display a “Message Waiting” or “Critical Message Waiting” icon to the upper left because all messages are presented at the bottom of this view.

The following conventions apply to interpreting the Monitor Chemistry View:

Area Description

Upper right corner

A system status indicator in the upper right corner.

Three Chemistry panes

Three Chemistry panes (Synthesis, Clv/Dep, Dep/Pur) occupying about one half of the window which present information about the chemistries in progress.

Small middle pane

A small middle pane which provides information about the sequences currently undergoing synthesis on the Synthesis pane.

Lower pane A lower pane which presents a series of messages from the instrument.

Convention Description

Grouping of three As shown in the figure above, the turntable position numbers are placed in the three panes in groups of three

Each group is a row Each group of three represents a radial row of column positions on the turntable which undergo the same type of chemistry.

Same cycle on each group

The instrument uses the same cycle on each group of three oligonucleotides and concurrently uses three different cycles at the processing stations.

Syn v4.20f Cleave v4.20g

, DB 4.20, 2.20.

, DB 4.20, 2.20.

C


The general status of the instrument is indicated in all views by presentation of one of the following four words in the upper right corner:

� Ready

� Running

� Manual Control

� Interrupted.

Three ChemistryPanes

The three chemistry panes (Synthesis, Clv/Dep, Dep/Pur) show details of the chemistries in progress in the three chemistry modules. After the first set of three columns reaches the Purification module, three chemistries are concurrently in progress on the instrument (until the end of the run).

Progress of Chemistry

Chemistry progresses as follows when a run is started:

Description of Chemistry Panes

All three panes show the same types of information, described in the two tables below:

Stage Action

1 First the Begin procedure can be seen executing in the left pane.

2 Next, when the first three columns are undergoing synthesis, only this type of chemistry is in progress and information is presented only in the left pane.

3 When the first three columns move to the Cleavage module, the next set of three oligonucleotides starts synthesis. Synthesis and Cleavage chemistries are concurrently in progress at this point.

4 Finally, when the first set of three columns reaches the Purification module, all three chemistries are concurrently in progress with all three panes showing chemistry details.

Location Purpose

Upper Part of Pane

This portion of each pane shows two types of information:

� The turntable position of each oligonucleotide, expressed as a number between 1 and 48, and

� The name of each sequence (Synthesis Order name; the name of the sequence may be different than the Synthesis Order name).

Note If no name is specified when making a Synthesis Order, the default name is the sequence name when one is entered.


Description ofSequence Pane

Information

The middle pane provides a listing for each of the three sequences currently being synthesized in the Synthesis module:

� The sequence on each row is identified by number as to the location of the column in the turntable.

� Each sequence is then listed with an orientation in the 5´ to 3´ direction.

� The numbers listed under the Base heading at the end of each row indicate the current base position being synthesized.

The number 2/25, for example, indicates that the second base position of a total of 25 bases in the sequence is currently being synthesized.

Note Following convention, a DNA sequence is entered 5´ to 3´. Likewise, the sequence listing in the ABI™ 3948 System displays each sequence in a 5´ to 3´ orientation. However, the DNA sequence is actually synthesized 3' to 5'. This is indicated in the Monitor Chemistry View by the highlighting of the current base position being synthesized.

Status and SystemMessages

The lower pane presents a series of messages from the instrument listing the times at which major processing events occurred. This listing may also include error and other system messages. The following messages are examples of system status messages. Each message is prefaced by the time at which an action was taken:

11:31:35 AM Starting Manual Control Action11:31:38 AM Manual Control Action Complete11:32:17 AM Starting Chemistry Run12:41:55 PM Interrupting system12:45:05 PM Resuming Chemistry

12:50:10 PM Chemistry Done

Location Name Description

Lower Part of Pane

Cycle Name Name of cycle in progress.

Step Number of cycle step currently in progress/total number of steps in the procedure.

SubStep/Loop

The standard chemistry cycles provided with the instrument use subroutines in executing chemistry.

SubSteps are steps in subroutines and, optionally, if present the Loop number specifies the current iteration of the current loop.

Fxn The name of the function currently being executed.

Fxn Time The remaining time that the function is active.

Listed for example as 15/20 where 20 is the total time the function is executed and the first number is a decrementing counter, which counts down from the second value to “0” upon completion of execution.

Fxn Number The number of the function. More information about functions is presented in this section (see “Manual Control View” on page 6-3) and Appendices A and C.


The log on the bottom of this view informs you of the following types of events:

� When an instrument run was interrupted or aborted

� When a sensor fails to detect liquid (which may or may not interrupt the run)

� Instructions used during procedures (such as autodilution)

� Power fail information

� Start and stop times of chemistry runs

� Execution of runs or manual control events

The information presented on this log is intended to inform a user about the normal progress of a run and for troubleshooting. For a full list of messages presented in the log, see "System Messages" on page C-38 of Appendix C

Using MonitorChemistry

Information

While information in the Monitor Chemistry view is primarily used to keep track of chemistry in progress, the log on the bottom also informs you when an interrupt or Pause After occurs so that you can perform a bottle change procedure, remove a high priority oligonucleotide, or extend the run.

Instructions to the user during procedures (calibration, bottle change, etc.) are presented in the log. You can also use the log for troubleshooting the instrument in conjunction with the Monitor Instrument view.


Monitor Instrument

Introduction The Monitor Instrument view, shown with a run in progress in the figure below, displays the status of system valves, liquid sensors, pressure regulators, as well as other parameters and instrument conditions. These parameters and conditions include

� Chemistry status

� Deprotection coil temperature

� Current turntable and sample collector positions

� Status of Synthesizer, Cleavage, and Purification jaws, and

� Status of heater and fan

This view is best used in conjunction with the Monitor Chemistry view to troubleshoot problems which may occur at any point during a particular chemistry.

Current Date andTime

The current ABI™ 3948 System date and time is displayed at the top of the window, to enable you to correlate particular events occurring in one of the chemistry cycles with the underlying hardware and/or software supporting chemistry.

Valves The Monitor Instrument view supports up to 80 numbered valves in its current form. The status of valves is indicated in groups of five symbols, 10 valves per line, with the status of a particular valve marked with either a bullet or an exclamation point:

� Valves that are off are indicated by a bullet, and

� Valves that are on are indicated by an exclamation point

Separating each line of 10 valves into two groups of five makes the display easier to read.


Liquid Sensors

Conventions The instrument has a number of liquid sensors placed in critical portions of its plumbing to ensure that liquid transfers are being made. See "Edit Cycle and Procedure Interface" on page 6-12 for information on enabling a sensor for a particular function.

When enabled in the cycle or procedure, a check in a liquid sensor check box indicates that liquid is detected at the current time at the sensor. In the discussion in this section, sensors are grouped according to:

� The position in the plumbing, or

� The type of phosphoramidite monitored

Except for the check boxes labeled A, G, C, and T, which indicate phosphoramidites, all of the letters A,B, and C included as part of a sensor designation indicate one of the three positions in a jaw mechanism:

You can locate the sensors by number on the instrument plumbing diagram provided in Appendix D. A checked box for a particular sensor means that liquid is present at the sensor at the current time. An unchecked box means that gas is present at the sensor at the current time.

Synthesis Jaw/ValveBlock/Manifold

Sensors

The synthesis sensors listed in the table below are in the plumbing on either the upper or lower side of the synthesis jaw mechanism:

� The first three sensors are located on the upper side of the jaw mechanism.

� The next three sensors, labeled “Man.”, are located just below the first manifold under the synthesis jaw.

� The last three sensors, labeled “Syn. Lower,” are located closest to the reagent delivery valve blocks.

Position Description

Position A Position A corresponds to the outer in a row of columns in the turntable.

Position B Position B is the middle position.

Position C Position C is the inner position.

Name Identity Purpose

SUA Syn. Upper A - Sen. 3 Determines that reagent for the listed position has reached the sensor position.SUB Syn. Upper B - Sen. 2

SUC Syn. Upper C - Sen. 1

ManA Man. A - Sen. 22 Determines that reagent for the listed position reaches the sensor position.ManB Man. B - Sen. 23

ManC Man. C - Sen. 24

SLA Syn. Lower A - Sen. 6 Determines that reagent for the listed position reaches the sensor position.SLB Syn. Lower B - Sen. 5

SLC Syn. Lower C - Sen. 4


Cleavage JawSensors

The three sensors listed in the table below are in the plumbing on the upper side of the cleavage jaw mechanism:

Deprotection CoilSensors

The three sensors listed in the table below are in the plumbing on the exit side of the deprotection coils:

Purification JawSensors

The three sensors listed below are in the plumbing on the upper side of the purification jaw mechanism:

PhosphoramiditeSensors

The four sensors listed below are in the plumbing between the phosphoramidite bottles and Valve Block B:

UV Detector Sensor A single sensor, labeled UVQ, is in the plumbing input to the UV detector.

Pressure Regulators The current values in psi (pounds per square inch) for the ten pressure regulators are shown in this portion of the Monitor Instrument view:

� The ten pressure regulators initially have the values listed in the table.

� Except for Regulators 1, 3 and 10, the regulators are maintained within ±0.2 psi of the initial pressures.


CA Cleavage A - Sen. 7 Determines that reagent for the listed position has reached the sensor position.CB Cleavage B - Sen. 8

CC Cleavage C - Sen. 9


DEPA Coil A - Sen. 10 Determines that reagent for the listed position has reached the sensor position.DEPB Coil B - Sen. 11

DEPC Coil C - Sen. 12


PA Pur. A - Sen. 14 Determines that reagent for the listed position has reached the sensor position.PB Pur. B - Sen. 15

PC Pur. C - Sen. 16


A Phos. A - Sen. 17 Determines that reagent for the listed position has reached the sensor position.G Phos. G - Sen. 18

C Phos. C - Sen. 19

T Phos. T - Sen. 20


� Regulators 1, 3 and 10 are programmed to vary pressures during the Cleavage and Purification cycles.

Other Parametersand Instrument

Conditions

The parameters and conditions listed under the Other category on the Monitor Instrument view are as follows:

� Deprotection coil temperature

� Opcodes

� Current turntable and sample collector positions

� Status of Synthesizer, Cleavage, and Purification jaws, and

� Status of heater and fan

Information for the parameters above is presented in the table below:

Reg. No. Pressure Reg. No. Pressure

1 10 2 8

3 10 4 7

5 8 6 6

7 10 8 9

9 6 10 10

Parameter Description

Temperature (Temp) The value presented for this parameter is the current temperature of the deprotection coils.

During the Cleavage and Purification cycles, the temperature of the coils is programmed using the Set Coil Temp parameter (Function 195), designating the new temperature with the MISC data entry for the function.

Opcodes Messages presented in this field are only of interest to system developers. No user level information is presented here.

TT Position This is the current synthesis module turntable position (1-18)

SC Position This is the current sample collector position (1-48)

SC Rack Type Indicates the type of rack in the sample collector, either Red 6x8 (standard OligoRack) or White 4x12 (screw top microtiter).

Syn Jaw This is the current status of the Synthesizer jaw mechanism, either Open or Closed.

Clv Jaw This is the current status of the Cleavage jaw mechanism, either Open or Closed.

Pur Jaw This is the current status of the Purification jaw mechanism, either Open or Closed.

Heater and FAN check boxes

If either of these check boxes is checked, the device associated with the check box is turned on.


Monitor Run View

Introduction The Monitor Run view, shown in the figure below, is another way of observing the progress of an ABI™ 3948 System run. Information in this view is useful in tracking the general course of chemistry, knowing when to set a Pause After in the run, and is a table of the entire run which provides useful information in tracking the run.

Types of InformationProvided

The Monitor Run view provides the following types of information about the run in the form of a table:

Type of Information Description

Row Each of the 16 rows in the turntable is labeled by number in the first column.

Column A Names of the three Synthesis Orders on each row of the turntable are listed row by row in second through fourth columns of the table.Column B

Column C

Chemistry Status The fifth column lists the current chemistry status for each row – a particular row has one of these notations:

� 1) Scheduled/Not scheduled

� 2) Synthesizing

� 3) Cleaving

� 4) Purifying

� 5) Completed

Pause After The sixth column, Pause After, has either “No”, “Yes”, or nothing after a row to indicate whether a pause has been programmed.


Section 2 - Using the Run Setup View and Extending a Run

Contents of Section 2 This section describes how to use the Run Setup view and extend a run.


Topic See page

Importing Synthesis Orders 5-21

Process of Importing Synthesis Orders 5-21

Description of Open Dialog Box Buttons 5-22

Selecting Orders for the Next Run 5-23

Description 5-23

Synthesis Order Information Provided for a Selected Sequence 5-24

Provided for a Selected Sequence 5-24

Assigning Chemistry and AutoSorting 5-25

Introduction 5-25

Assigning Chemistry 5-25

AutoSorting 5-25

Bottle Usage 5-27

Procedure 5-27

Using the Load Display 5-28

Types of Information Provided 5-28

Procedure for the Load Display 5-28

Significance of the Three Positions 5-28

Protocol and Cycle Identification 5-29

Using the Buttons on the Display 5-29

Extending a Run 5-30

Using Pause After 5-30


Importing Synthesis Orders

Process of ImportingSynthesis Orders

This subsection lists the process of importing Synthesis Orders into the Run Setup view and provides detailed information about the Open dialog box used for importing.

The Open button on the Run Setup view is initially the only button active in the Synthesizer window. When it is clicked, the dialog box below is presented to enable entry of Synthesis Orders into the Run setup view:

Figure 10-1 Import Synthesis Order Dialog Box

The dialog box in Figure 10-1 is used as follows:

Stage Description

1 Synthesis Orders are accessed in the usual way in the top scroll box.

2 Once orders are present in the top scroll box, the orders to used in the next run are moved to the bottom scroll box by clicking the Add or Add All buttons.

The Add button moves only those orders selected while the Add All moves all orders from the top to the bottom scroll box.

3 Any orders placed in the bottom scroll box by mistake can be removed using the Remove button.


For the procedure on using the dialog box as outlined above, see "Assigning to the Run Setup View" on page 2-20 of the ABI™ 3948 System User’s manual.

Description of OpenDialog Box Buttons

The Open dialog box is used as described in the table below to load sequences into the Sequence Order list (see the lower figure on page 5-9).

4 When the set of orders to be input to the Run Setup view is present in the lower scroll box, click the Done button to move them to the Sequence Order list, the top scroll box in the Run Setup view. as shown below:

The dialog box in Figure 10-1 is used as follows: (continued)

Stage Description

Dialog Box Button or Checkbox Description

Add/Add All One of these buttons is used to move Synthesis Orders from the upper scroll box to the lower scroll box in the dialog box. The Add button moves selected Synthesis Orders and the Add All button moves all orders to the lower scroll box when a single order is selected.

Show all Files Checkbox

Click the check box labeled Show all files to view all file names, regardless of the file’s format.

Remove If you have inadvertently moved one or more Synthesis Orders to the lower scroll box, this button moves selected orders back to the upper box.

Eject If you are adding Synthesis Orders from a diskette in the floppy drive, this button enables you to eject a floppy so that you can insert another.

Done When you have finished moving Synthesis Orders to the lower scroll box, click Done to close the dialog box and transfer all orders in the lower scroll box to the list of Ordered Sequences in the Run Setup view.

Cancel This button enables you to return to the Run Setup window without adding sequences.


Selecting Orders for the Next Run

Description The next step in inputting Synthesis Orders for a run is to move the orders to be produced by the next run from the Sequence Order List (at the top of the Run Setup view) to the Run Setup list at the bottom.

This is done by selecting the orders in the top list and then clicking the Add+ button to move them to the lower list. For the procedure on this, see "Assigning Sequences for the Run" on page 2-22 of the ABI™ 3948 System User’s manual.


Synthesis Order Information

Provided for aSelected Sequence

As shown in the figure below, whenever a single sequence is selected in the Run Setup scroll box, several types of information are presented for that sequence below the scroll box. This information includes:

� A detailed base by base listing between the 5´ and 3´ ends

� The Specification that oligonucleotide be

– Purified, or

– Crude

� The length of the sequence

Note If you decide not to produce a particular sequence during the upcoming run, click Remove to return it to the upper list.


Assigning Chemistry and AutoSorting

Introduction Once the desired sequences have been moved to the Run Setup scroll box, the next thing to do in preparing for a run is to assign chemistry and then autosort, if necessary, to determine turntable loading positions.

Assigning Chemistry The general process of chemistry needed to produce an oligonucleotide is described in the following table:

AutoSorting Do the following to autosort sequences:

Chemistry Description

Protocol Select the sequence then choose the appropriate protocol from the Protocol pop-up menu to make an assignment.

The following rules apply to assigning protocols:

� All sequences in a given row must have the same protocol assigned to them.

� A protocol must be assigned to each sequence in the Run Setup scroll box.

� A protocol assignment can be made for Multiple orders by using the shift key to select them and then select the protocol.

� To assign a single protocol to all sequences, select a single sequence and use Command-A (�-A) before choosing the protocol.

The word Protocol appears next to a pop-up menu of protocols that are listed in the Run Protocol view (see Run Protocol View). You must assign a protocol to each sequence in the Run Setup scroll box.

Begin Procedure Use the default choice unless you have developed an additional Begin procedure you want to use.

This pop-up menu lists the pre-defined Begin procedure as well as possible additional undefined procedures. Use the Edit Begin Procedure view to define a new Begin Procedure (see page 6-20).

End Procedure Use the default choice unless you have developed an additional End procedure you want to use. This pop-up menu lists one standard End procedure and possible additional undefined procedures. Use the Edit End Procedure view to define a new End Procedure (see page 6-21).

Stage Description

1 Use the AutoSort button to sort the sequences in the Run Setup Order box.

Note The AutoSort button does not become active until each of the sequences in the Run Setup Order has an assigned protocol.

The top row of sequences in the Run Setup Order box is the first row of sequences synthesized.


2 AutoSort applies the following sorting rules to guarantee that any set of oligo orders, once sorted, resort to the same positions:

� First, sequences are arranged in rows by protocol.

� Second, sequences are arranged by the number of bases in each sequence.

The longest sequence in a row is on the left; the shortest sequence in a row is on the right.

� Third, sequences are arranged according to the 3´ end base, with A before C, C before G, and G before T.

If two or more sequences in a row contain the same number of bases AND the same 3´ base, the sequence that contains an A at the 3´ position goes before a sequence containing a G at the 5´ end, and so on.

� Fourth, sequences are arranged by their sequence order file (Macintosh file) names.

Note

3 To change the position of a single sequence for any reason, the Arrow buttons are used as described below:

Stage Description

Step Action

a. Select a sequence by clicking it once.

b. Click an arrow once to move the sequence one location. You can click any arrow repeatedly or click any combination of arrows


Bottle Usage

Procedure The Bottle Usage button becomes active after protocols have been assigned to the sequences in the Run Setup Order scroll box:

Step Action

1 Click Bottle Usage to see the display of reagent requirements for the syntheses ordered, like that shown below:

The calculations for required volumes are based on the data found on the Reagent Utilization Table (see page 6-9). Compare the volumes required for each reagent shown in this Bottle Usage display to the actual amounts of each reagent attached to the instrument.

2 Compare the volumes required for each reagent shown in this Bottle Usage display to the actual amounts of each reagent attached to the instrument.

3 To return to the original Run Setup view, click OK.

31


Using the Load Display

Types of InformationProvided

This subsection provides the following information:

� When to Use the Load Display

� Significance of the Three Positions

� Protocol and Cycle Identification

� How to Use the Buttons on the Display

Procedure for theLoad Display

Do the following to use the load display:

Significance of theThree Positions

This Run Setup view displays one row of three oligonucleotide positions on the turntable at a time:

� Each position is represented by a circle with a number.

� Each colored circle contains a colored letter that corresponds to the type of OneStep column required at each position: green=A, yellow=G, red=C, and blue=T.

� Each type of column provides the support required for the initial base at the 3' position.

Use this view as a map to load columns into the turntable.

Step Action

1 Click the Load button (figures on page 5-24) when you are ready to load OneStep columns on to the ABI 3948 System turntable.

2 After you click this button, the Run Setup view changes to display a wedge-shaped area on the turntable (like that shown below):


Protocol and CycleIdentification

The name of the protocol and chemistry cycles assigned to the three oligonucleotides on the current turntable row are listed at the bottom of the view. This information is useful when using the Scan function in this view (Start Scan button) to double check your assignment of Protocol with its included set of Synthesis, Cleavage, and Deprotection cycles.

Using the Buttons onthe Display

The buttons in the Load Display are used as described in the table below:

Button Description

Next Click the Next button to advance to the next row of columns in the turntable. Each click moves the turntable one row and updates the view.

Note The front panel “Column load” button on the instrument has the same affect as the Next button

Prev Click the Prev button to move the turntable in the reverse direction. Each click moves the turntable a single row and updates the view

Start Scan Click the Start Scan button to quickly look at all of the positions on the turntable and visually check that the proper column was loaded into the position.

The scan displays all columns before stopping, pausing briefly at each row. Stop Scan terminates the turntable scan.

Cancel Click the Cancel button to return to the original Run Setup Order view without starting the instrument.

Note If you want to view the Bottle Usage display, you must click the Cancel button to return to the Run Setup Order.

Start Click the Start button to begin instrument operation.

After you click Start, the turntable rotates to the starting position and the information about sequences, protocols, and column positions is transferred from the computer to the instrument.

Note A status indicator is presented to indicate that information is downloading.

As soon as loading is complete, the Synthesizer window view changes to Monitor Chemistry, and synthesis is initiated.

Refer to the manual section entitled “Monitor Chemistry View” for information on monitoring chemistry during synthesis. If you desire to monitor hardware conditions occurring during synthesis, change to the Monitor Instrument view (see page 5-15). The Monitor Run view shows the overall progress of the instrument through the run.


Extending a Run

Using Pause After The column of check boxes on the right side of the Run Setup Order scroll box, labeled Pause after, is used to indicate when a pause is programmed using the Pause After command (Synthesizer menu). When an X appears in a box, an instrument pause occurs when that row of sequences has been synthesized.

Note You can only program a Pause After once a run has started.

Pause After is used to extend a run (see "Extending a Run" on page 2-35 of the ABI™ 3948 System User’s manual).

IMPORTANT Only a Pause After, not an interrupt, may be used to extend a run.

For information on setting a Pause After, see “Pause After Command” on page 4-23.

For more information on run extension, see “Extending a Run” on page 2-35 of the ABI 3948 User’s Manual.

Synthesizer Views Supporting Operation 6-1

Synthesizer Views Supporting Operation 6

In This Chapter

Topics Covered This chapter provides information on the Synthesizer Views not covered in Chapter 5, which support the run but are not necessarily accessed during the run. The various Edit Cycle and Procedure views used to support chemistry are discussed in Section 2 of this chapter.


Section See page

Non-Cycle Operational Support Views 6-2

Editing Cycle and Procedure Views 6-11

6

6-2 Synthesizer Views Supporting Operation

Section 1 – Non-Cycle Operational Support Views:

Topics Covered This chapter describes the Synthesizer Window views used to support instrument operation and covers the following topics:

Topic See page

Manual Control View 6-3

Description of View 6-3

Types of Functions 6-4

Viewing a Function Valve List/Creating a User Function 6-4

Creating a User Function 6-5

Printing Functions 6-5

Power Fail View 6-6

Purpose of View 6-6

Instrument Test View 6-7

Tests Performed 6-7

Activating Tests 6-7

Interpreting Tests 6-7

B+ Tet Calibration 6-9

Contents of View 6-9

Reagent Utilization Table 6-10

Contents of View 6-10


Manual Control View

Description of View The Manual Control view is used to exercise manual control over existing functions and create or edit user functions. When first selected, the Manual Control view appears as shown below:

Two kinds of functions are used in the instrument, system functions and user functions:

� System functions are permanent dedicated functions provided with the synthesizer and cannot be edited.

� User functions, listed in the table below, can be both viewed and edited.

Selecting a user function in the Function List (shown below) enables the CHOOSE VALVE TO ADD scroll box enabling entry.

Table 5-4 User Functions

110

150

227

228

380-394

401-500

Ready


Types of Functions Functions are of several types:

� Valve functions

� Non-valve hardware functions

� Logical or cycle directive functions

Valve functions, for example, simultaneously open a series of valves to perform a defined action during synthesis when executed from a cycle. The valves opened as part of a function remain open for the time specified in the cycle step or otherwise as controlled by software. See Appendix A for a complete listing and discussion of functions.

Areas of the ManualControl View

The Manual Control view provides three scrollable lists and a fourth area which allows turning on a function:

� The FUNCTION LIST indicates which function is currently displayed.

� The VALVES OPEN FOR FUNCTION list contains a valve listing for the current function, if that function opens valves.

� The CHOOSE VALVE TO ADD buttons are used to create user functions and contains a list of valves available for creating a new function.

� The fourth area enables manual control and is described in the procedure immediately below.

Exercising ManualControl of a

Function

The area on the upper left labeled Manual Control, allows you to turn on a selected function for a specified period of time and provides the option of using the liquid sensors with reagents delivered by the function. To use this portion of the table, follow these steps:

Viewing a FunctionValve List

To view the valve list associated with an existing function:

� Select the function to be viewed in the FUNCTION LIST, which contains a complete list of functions contained in the instrument.

Selecting a function on this list displays the list of associated valve openings (no valves are used in some functions) in the list entitled VALVES OPEN FOR FUNCTION and lists the number and name of the selected function in the two fields above the list (Function and Name).

Step Action

1 Select the function to be turned on in the Function List.

2 Enter the time during which the function operates in the Time entry field (optional for certain functions).

3 For functions which require a Misc entry, such as Functions 301–310 (Set Pres Reg), enter the appropriate value in this field.

4 Click Start to turn on the selected function for the specified time, if applicable.

5 If you want to stop the function before the time specified has elapsed, click Stop.


Creating a UserFunction

To create a user function, do the following:

Printing Functions The Manual Control View is one of several views from which printing can be done (others are the Edit Cycle views, the Edit Procedure views, and the Instrument Test view). As shown in the figure below, the print dialog box for the Manual Control view allows two main options, printing of function text or printing of the current Manual Control view window image.

If you choose the Text option, you can choose to print a single selected function, system or user, or choose to print all user functions. The function number and name are printed for all functions but valve numbers are printed only for system or user functions with associated valves. Functions with no associated valves (system or empty user functions) will have “None” printed for VALVES.

Note The User functions available are listed in the table on page 6-4.

Step Action

1 Select any of the functions in the list entitled “User Fxn” (see available numbers in the table on page 6-3) or functions in the range 401–500 (for user functions tied to sensors). (The function number will appear in the Function field and the Name field will become available for entry.)

Note Selecting an empty user function displays the function number in the Function field and the function name in the Name field. Nothing is listed in the VALVES OPEN list for an empty function or a non-value function.

2 Type a name for the new user function into the Name field.

3 Select the first valve to be added from the CHOOSE VALVE TO ADD list and then click the Add button to add the valve to the VALVES OPEN FOR FUNCTION list.

The CHOOSE VALVE TO ADD list is a complete list of valves used to create user functions.

4 Continue by selecting each valve to be added to the function in the correct order and then clicking the Add button. If you make an error, click the Remove button.


Power Fail View

Purpose of View The Power Fail view, shown below, is used to review the power failure history of the synthesizer.


Instrument Test View

The Instrument Test view pictured below is primarily intended for Service Engineer use. All tests in this view may be performed by the customer but interpretation of time, current, and valve status values is “service only.” If an explanation of these values becomes necessary, please ask your local field representative for interpretation.

Sensor status is intended for use by the customer and the service engineer. Once a sensor calibration is performed (in the Misc Procedures view), gas and liquid values are assigned for each of the twenty-three (there is no sensor #13) liquid sensors. The liquid value must be at least twice the gas value for each position in order for the calibration procedure to pass and for a Synthesis to start. Your local engineer or technical support representative may ask you to read off the sensor values when troubleshooting failed deliveries.

Note The Instrument Test View is one of several views from which printing can be done (others are the Edit Cycle views, the Edit Procedure views, and the Manual Control View.


B+ Tet Calibration

Contents of View The figure below contains the initial B+ Tet calibration values for your 3948 instrument. If this table is deleted or otherwise damaged in your instrument, it can be restored in one of two ways:

� Open the off-line copy of the database and then download the database using the Send to Synthesizer command.

� Open the off-line copy of the database and then use the opened database as a reference while you manually type in appropriate values.

Manual entry is made one field at time into the table in the ABI™ 3948 System Control application. Select each table field, i.e. B+TET to Col A, in a particular column and then enter the appropriate value in the time entry field at the bottom of the table.

The values provided in this table are used by the B+Tet function in the Synthesis module to provide a maximum time for delivering amidite from a bottle to a column position. For example, the Tet to Col value is the time (in seconds) for the washout with tetrazole of the synthesis valve block to ensure that there is no crossover amidite delivery between columns.

For more information on how the B+Tet function works, see the information in Appendix B, ABI 3948 Special Functions, under “1 B+TET to Syns” on page B-5.

Note The CPL (Coupling) Wait Time in the cycle waits between pushes during the coupling phase of synthesis.


Reagent Utilization Table View

Contents of View The figure below contains the initial Reagent utilization values for your ABI™ 3948 instrument. If this table is deleted or otherwise damaged in your instrument, it can be restored in one of two ways:

� Open the off-line copy of the database and then download the database using the Send to Synthesizer command.

� Open the off-line copy of the database and then use the opened database as a reference while you manually type in appropriate values.

Manual entry is made one field at time into the table in the ABI™ 3948 System Control application. Select each table field in a particular column and then enter the appropriate value in the volume entry field at the bottom of the table.

The values provided by this table are used to calibrate the Bottle Usage View so that the latter view can correctly indicate the amounts of reagents needed for the next run.

The volume values in the four columns are described in the following table:

Column Description

Base Volume consumed per addition of the specified base (from Positions AGCT and 5–8).

Cycle Volume consumed per synthesis cycle of specified reagents other than amidites.

Example: for a 20 mer oligo, total TCA consumption with be (20 x value in Cycle column) or 20 x 0.58 = 116 mL for that oligo.


Oligo Volume consumed of specified reagent on a per-oligo basis.

Example: for a run of 36 purified oligos,total consumption of TEAA will be1.01 x 36 = 36.36 mL for the entire run (1.01 mL per oligo).

Run Consumption of reagents used only once per run.

Column Description


Section 2 – Editing Cycle and Procedure Views

Topics Covered This section contains the following topics:

Edit Cycle and Procedure Interface 6-12

Seven Editing Views 6-12

Interface Elements 6-12

Same General Layout 6-12

Cycle and Procedure Pop-Up Menus 6-13

Buttons 6-14

Entry Fields 6-16

Edit Synthesis Cycle View 6-17

Purpose and Contents of View 6-17

Edit Cleavage Cycle View 6-18


Edit Purification Cycle View 6-19


Edit Begin Procedure View 6-20


Edit End Procedure View 6-21


Edit Bottle Procedure View 6-22


Misc (Miscellaneous) Procedures View 6-23



Edit Cycle and Procedure Interface

Seven Editing Views Seven views are available for editing existing procedures or cycles and creating new procedures or cycles:

� Edit Synthesis Cycle

� Edit Cleavage Cycle

� Edit Purification Cycle

� Edit Begin Procedure

� Edit End Procedure

� Edit Bottle Procedure

� Misc (Miscellaneous) Procedures

Interface Elements All seven editing views share the same interface elements:

� Pop-up menus

� Scroll boxes

� Buttons

� Entry fields, and

For a detailed procedure on how to use Edit views, see Section 6 in the ABI™User’s Manual (Advanced Use of 3948 System Control).

Note The Edit Cycle, Edit Procedure, Manual Control, and Instrument Test views are the only views which can be printed.

Same GeneralLayout

All Edit views have the same general layout as that shown for the Edit Cycle view example below. Refer to this figure in following the descriptions of the editor interface which follow:

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Cycle and ProcedurePop-Up Menus

A pop-up menu displays available cycles or procedures to edit and presents the choices available for the particular view. For example, the Cycle menu on the Edit Synthesis Cycle view initially presents the pop-up menu shown below:

When you select one of the names on this type of menu, the contents of the cycle or procedure are listed in the main scroll box and the name is presented in the Name entry field. If you select a name representing a new cycle or procedure, the main scroll box initially has only Begin and End steps as shown in the figure below:


Buttons The following buttons are described in this subsection:

� Apply

� Copy from

� Execute

� Insert

� Safe/Sensor

Apply Button

Clicking the Apply Button updates the cycle with all the information entered into the previous fields. If there is an invalid entry, the Apply button will be grayed out until all the fields are correct.

Copy from Button

Once an empty cycle/procedure is selected, the Copy from Button becomes available:

� This button displays a dialog box like that shown below:

� Use the dialog box to copy any existing cycle from any open database as a template for creating a new cycle or procedure.

Initially, the dialog box shows only the standard cycle provided.

Note If you access a database containing several types of cycles or procedures, the dialog box shown above will only show the cycles or procedures which are relevant for the current type of edit view.

Execute Button

You can execute Begin, End, Bottle, and Shutdown procedures whenever the instrument is “Ready”. The button is grayed out to disable execution when the instrument is in any other mode.

In the Edit views for Synthesis, Cleavage/ deprotection, and Purification cycles, the Execute button is always grayed out to indicate that you cannot manually execute these cycles. The cycles are executed by the chemistry protocol assigned for instrument operation.

Dbase 4.20, 2.20: Syn v4.20


Insert Button

This button is used to insert new blank steps into the cycle listed to the right.

Safe/Sensor Buttons

These radio buttons allow you to designate, respectively, that a new step is not a safe step and that sensors are used for the step. (As defaults, a step is automatically designated as a safe step which does not use sensors.) Entries made with these buttons appear in the SAFE and SNS columns of the cycle table.

Only certain functions use liquid sensors as part of their operation.

Step Action

1 Select the step above your new step.

2 Click Insert.

As soon as Insert is clicked, a new blank line is added to the cycle or procedure listing and the cursor moves to the Function entry field

3 Select the function in one of two ways:

� Type in the function number in the Function field.

� Click the function you want to insert from the Function scroll box, shown below:

Note Clicking a function is often easier than typing in the function number.

As soon as a function is added as a step in the main scroll box, the new step is open for input using the remaining two entry fields, Time and Misc, and the two radio buttons, Safe and Sensor.


Entry Fields The following entry fields are described in this subsection:

� Function Entry field (Function Scroll Box)

� Misc Data Entry field

� Name Entry field

� Time Entry field

Function Entry Field/Function Scroll Box

The Function Entry field is used to add a function to new blank steps placed in the cycle or procedure in the main scroll box with the Insert Button. It requires that you know the associated number by heart.

The Function scroll box, shown in the figure on page 6-15, is an easier way to add a function to new blank steps since you can scroll until you identify the proper function.

Misc Data Entry Field

For some types of functions, an entry is required for this field to make an entry in the MISC column of the cycle table. For example, a loop index or a pressure setting can go in the MISC field. Refer to “Special Logical Functions” in Appendix A for information on how to set parameters for special functions.

Note If the Misc field is blank, the Apply button will remain grayed out (unavailable). All such fields require at least a zero.

Name Entry Field

This field displays the current name of the cycle selected from the Cycle menu. It initially has the name selected, as indicated by highlighting, allowing you to change the name.

Time Entry Field

After you have inserted a function as a step in the cycle, you need to enter the time for the new step in this entry field. Select the default “0.0” entry and type in the new value to enter it into the cycle table.


Edit Synthesis Cycle View

Purpose andContents of View

The Edit Synthesis Cycle view allows you to produce custom cycles to perform synthesis in the Synthesis module:

� The Edit Synthesis Cycle view initially appears as shown in the left side of the figure below.

� The Cycle pop-up menu, shown in the right side of the figure, is used to select an existing cycle or select a location for creating a new cycle.

The Synthesis Cycle pop-up menu can store up to 20 cycles. The first cycle is a permanent non-programmable cycle for use in the instrument. You can copy it into one of the other 19 locations labeled “Syn Cycle” and then edit it to create a custom cycle.

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Edit Cleavage Cycle View


The Edit Cleavage Cycle view allows you to produce custom cycles to perform cleavage at the Cleavage station and deprotection in the deprotection coils:

� The Edit Cleavage Cycle view initially appears as shown in the left side of the figure below.


The Cleavage Cycle pop-up menu can store up to 9 cycles besides the first cycle (which is a permanent non-programmable cycle). You can copy it into one of the other 9 locations labeled “Clv Cycle” and then edit it to create a custom cycle.

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Edit Purification Cycle View


The Edit Purification Cycle view allows you to produce custom cycles to perform purification at the Purification station:

� The Edit Purification Cycle view initially appears as shown in the left side of the figure below.


The Purification Cycle pop-up menu can store up to 10 cycles. The first three cycles are permanent non-programmable cycles for use in the instrument:

� The first cycle is used for purification of regular oligonucleotides.

� The second cycle is used for purification of dye primer oligonucleotides.

� The third cycle is used for purification of biotin.

You can use any of these cycles “as is” or you can copy one of them into one of the 7 locations labeled “Pur Cycle” and then edit it to create a custom cycle.

x.xx

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Edit Begin Procedure View


The Begin procedure provided with the instrument is a phosphoramidite purge procedure that fills all phosphoramidite and tetrazole delivery lines (from the reservoir to the reagent valve block) with fresh reagent. You should use the begin procedure prior to beginning a run or if one of the phosphoramidite reservoirs has not been accessed within 12 hours.

The Edit Begin Procedure View allows you to produce custom Begin procedures to prepare for a run:

� The Edit Begin Procedure view initially appears as shown in the left side of the figure below.

� The Cycle pop-up menu, shown in the right side of the figure below, is used to select an existing begin procedure or select a location for creating a new procedure.

The application can store up to 9 procedures besides the permanent non- programmable procedure provided. You can use the Begin procedure “as is” or you can copy it into one of the other 9 locations labeled “Beg Proc” and then edit it to create a custom procedure.

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Edit End Procedure View


An End procedure, shown below, is used to flush and clean out the system at the end of a run.

� The Edit End Procedure view initially appears as shown in the left side of the figure below.

� The End Procedure popup menu, shown in the right side of the figure, is used to select an existing cycle or select a location for creating a new cycle.

The application can store up to 9 procedures besides the standard end procedure provided. The standard end procedure is a permanent non-programmable cycle. You can use this procedure “as is” or you can copy it into one of the other 9 locations labeled “End Proc” and then edit it to create a custom procedure.

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Edit Bottle Procedure View


Bottle change procedures are used to autodilute amidites or remove empty bottles and replace them with bottles of fresh reagents. These procedures are used either before beginning a synthesis or when an active synthesis has been interrupted. Change procedures are especially important for phosphoramidite and tetrazole bottles because they are the most sensitive to atmospheric oxygen and water.

As shown in the Procedure pop-up menu in the right side of the figure above, fifteen non-programmable procedures are provided. The first five procedures, autodilution procedures for amidites, include:

� The “AutodiluteACCT” procedure which autodilutes amidites simultaneously at all four standard positions (positions 1 through 4)

� A separate autodilution procedure for each of the four standard amidite positions.

The next ten procedures, bottle change procedures, include:

� Procedures for use in changing the bottles at the four standard positions (positions 1 through 4)

� Procedures for ammonia and tetrazole bottles

� Procedures for manually diluted monomers in positions 5 through 8.

The user-defined procedures, labeled “Bottle Proc.,” are for use in creating a modified form of one of the standard procedures.

x.xxx.xxx.xxx.xx

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x.xx)

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Ready


Misc (Miscellaneous) Procedures View


This view contains various procedures needed for system maintenance or to shut down the instrument to prepare for long-term storage (storage for periods of one month or longer). Procedures such as “Clean Cols” and “Clean Lines” are useful for both maintenance or shutdown since they remove all reagents in the delivery lines and wash and dry all chemical pathways.

One of the procedures provided with the synthesizer, “Prime Amidites”, is the initial Misc Procedures View and is used to prime phosphoramidite delivery lines in the instrument after using the cleaning procedures or when restoring a shut down instrument for regular use.

Note Hardware Test vx.xx is a procedure used to implement hardware tests for the Instrument Test View and can not be executed from the Misc Procedures View even though the Execute button is active.

As you can see by examining the Cycle pop-up menu, the following types of procedures are permanent non-programmable cycles for use in the instrument:

� Procedures for cleaning, priming and hardware testing

� Procedures for calibrating sensors, verifying sensor calibration, and setting pressures

� Procedures for emptying ACN lines, and testing the sample collector and pressure module

Note The Janitor procedure provides more complete instrument cleanup.

You can use these procedure “as is” or you can copy one of them into an empty procedure slot and then edit it to create a custom procedure.

Note For information on shutting down the instrument, refer to “Shutting Down and Restoring the Instrument” on page 5-5 of the ABI ™ 3948 System User’s Manual.

x.xx

x.xxx.xxx.xx

,Ready

x.xx

x.xxx.xx

x.xx

x.xxx.xx

x.xx

x.xx

x.xx

Valves and Functions A-1

Valves and Functions A

In This Appendix

Topics Covered This appendix contains the basic information needed to understand ABI 3948 System Functions, Cycles, and Procedures. For detailed information on ABI 3948 System Special Functions, see Appendix B. For detailed information on ABI™ 3948 System cycles and procedures, see Appendix C. For a detailed listing of ABI™3948 System function information, see Appendix D. Appendix E provides a plumbing diagram for use in tracing function, cycle, and procedure reagent flow paths.

This appendix covers the following topics.

Topic See page

Information on ABI 3948 System Chemistry Terminology A-3

Introduction A-3

Definition of Functions, Steps, Cycles, Protocols, and Procedures A-3

Chemistry Stages on the ABI 3948 System A-3

Valves A-5

List of Valves A-5

Overview of Functions A-7

Introduction A-7

Function Names A-7

Time and Misc Field Entries or Subroutines A-7

Tracing Flow Paths for Functions A-8

Valve Functions A-10

Types of Valve Functions A-10

Uses of Valve Functions A-10

Manual Control A-10

Creating Your Own Functions A-10

Sensors A-11

General Characteristics A-11

Location of Sensors A-11

Retries A-12

Description of User Functions A-12

Components of Sensor-Controlled User Functions A-12

Names and Locations of Sensors A-13

A

A-2 Valves and Functions

Use of Parallel with Serial User Functions A-13

Sensor-Controlled User Function Example A-14

Valve Functions by Functional Category A-15

List of Function Categories A-15

Synthesis Valve Functions A-15

Synthesis Column Flushing and Back Flushing Functions A-16

Cleavage Functions A-17

Purification and Quantitation Functions A-18

Bottle Change Functions A-20

User Functions A-20

Block Functions A-21

Non-Valve Hardware Functions A-23

Overview A-23

Pressure Regulating Functions A-23

Calibration Functions A-23

Turntable Control Functions A-23

Miscellaneous Hardware Functions A-24

Jaw Functions A-24

Sample Collector Functions A-24

Overview of Special Functions A-25

Introduction A-25

Topic See page


Information on ABI 3948 System Chemistry Terminology

Introduction This section contains information essential to understanding functions, the three types of cycles (Synthesis, Cleavage/Deprotection, and Purification) and procedures.

Automated production of oligonucleotides on the ABI™ 3948 System requires chemical deliveries to specified destinations on the instrument. These deliveries are controlled by electrically activating valves that open and close, creating various pathways through the ABI™ 3948 System.

Definition ofFunctions, Steps,

Cycles, Protocols,and Procedures

Each valve is assigned a number that can be used to open or close it. Most functions consist of a valve or set of valves that open to perform a specific delivery or task.

For example, Function 27, Cap to Syn Cols, delivers capping solution simultaneously to all three synthesis columns. To accomplish this, Function 27 opens valves 13, 14, 15, 16, 17, 18, 22, 24, 25, and 26 simultaneously. Other functions control hardware systems such as the turntable or control the flow of cycle execution.

A function is an individual action which may have Time and Misc (Miscellaneous) fields. Functions can be activated manually in the Manual Control view or may be programmed as steps in cycles (for example, Function 27 is activated for 15 seconds in the standard synthesis cycle).

A cycle contains a series of steps that perform a chemical process. The ABI™ 3948 uses three types of cycles to produce oligonucleotides:

� Synthesis cycles, to add bases

� Cleavage/Deprotection cycles, to cleave oligonucleotides from column supports and then deprotect them

� Purification cycles, to purify and quantitate or produce crude oligonucleotides for output to the sample collector

A protocol consists of three cycles and contains all the chemical steps needed to produce an oligonucleotide.

A Procedure is a series of steps that completes a task, such as changing a reagent bottle. Upon command, the steps are performed once and are not repeated. There are four types of operating procedures: begin, end, bottle and miscellaneous.

Chemistry Stages onthe 3948

The Synthesis, Cleavage/Deprotection, and Purification cycles are performed for each oligonucleotide:

Stage Description

1 First, the Synthesis cycle synthesizes the oligonucleotides in the first row on OneStep™ columns at the Synthesis jaw.

2 At the second stage, the turntable moves the oligonucleotide-containing columns to the Cleavage jaw.

The Cleavage/Deprotection cycle cleaves the oligonucleotides from the OneStep column in the jaw and transfers them to the deprotection coils to be deprotected.

3 In the third stage, the columns move to the Purification jaw where deprotection is completed and the deprotected oligonucleotides are purified or collected as crude product.


Every time one row of columns moves to the next station, the subsequent row of columns moves to the station just vacated. As the first row of oligonucleotides is being cleaved at the second station, the second row of oligonucleotides is undergoing synthesis at the first station.

When the second row in turn moves to the second station, the third row of columns moves to the first station, and so on. This concurrent use of three stations with three different, sequential cycles executing in parallel enables the high throughput of the ABI 3948 Synthesis/Purification system.


Valves

List of Valves During a synthesis, valves are automatically opened to create chemical pathways and allow all necessary reagent and gas deliveries. You can also manually operate valves in the Manual Control View before or after a synthesis or when a synthesis has been interrupted.

Table A-1 lists all valves with brief descriptions of the associated gas or reagent flow. Appendix D provides more detailed schematic information. Refer to “Delivery Valve Blocks” in Chapter 2 for a physical description of the valves.

Note To identify reagent valve blocks, see the plumbing diagram for the instrument in Appendix D.

Table A-1 Valves with Associated Reagents and Gas Flows

Block or Valve Type

Valve Reagent or Gas Flow Block or Valve Type

Valve Reagent or Gas Flow

A 12345678

ACN to SynGas to SynXfr from SynTetrazole“5” amidite“6” amidite“7” amidite“8” amidite

E 2930313233343536

Gas to Clv LowerXfr to Clv LowerH2O to Clv Lower

AmmoniaClv Col C LowerClv Col B LowerClv Col A LowerUnused Valve 36

B 910111213141516

“A” amidite“G” amidite“C” amidite“T” amiditeSyn Col C Lower Syn Col B LowerSyn Col A LowerXfr to Syn Low

F 37383940414243

Gas to Dep LowerACN to Dep LowerXfr Clv to DepCoil A Input Coil B Input Coil C Input Dep Low to Waste

C 1718192021

NMIAc20TCAIodineSyn Low to Waste

G 44454647484950

Gas to Dep UpperH2O to Dep Upper

Coil A Exit Coil B Exit Coil C Exit Xfr from DepDep Upr to Waste

D 22232425262728

Syn Upr to WasteSyn Upr Hal WasteSynthesis Col C UpperSynthesis Col B UpperSynthesis Col A UpperPMS VentGas to Syn Upper

H 5152535455

Gas to PurACN to PurTEAA TFA Ac Acid


I 5657585960616263

H2O to Pur

20% ACNXfr from DepPur Col CPur Col B Pur Col AXfr to UVPur Low to Waste

J 6465666768697071727374757677787980

Unused Valve 64Pur Col C UpperPur Col B Upper Pur Col A Upper Pur Upr to WasteGas to Pur UpperXfr to Hal WasteUnused Valve 71Waste Valve 72Unused Valve 73Ammonia VentGas to Clv UpperAmidite VentUV LowerGas to UVUV to WasteUnused Valve 80

Table A-1 Valves with Associated Reagents and Gas Flows (continued)

Block or Valve Type

Valve Reagent or Gas Flow Block or Valve Type

Valve Reagent or Gas Flow


Overview of Functions

Introduction The permanent non-programmable functions available on the instrument are considered “System” functions to distinquish them from another function category provided on the instrument, “User” functions. More information on User functions is provided below and later on in this appendix.

System Functions

There are three fundamental types of functions on the ABI 3948 System:

� Valve functions that deliver reagents and gas throughout the system

� Non-valve hardware functions that control parts of the ABI 3948 System such as the turntable, pressure regulators, or sample collector

� Logical or cycle directive functions such as Begin Loop, Select Pur Cols, or If Cyc Greater.

Logical functions help to define the behavior of other functions or affect the flow of control through the steps in a cycle

Each of the three types of functions above are covered separately as sections in this appendix.

User Functions

Two types of User functions are provided on the instrument. One type is considered a general purpose valve function and the other type is a special User function that can perform sensor-controlled deliveries. Information on creating these types of User functions is provided in Chapter 6 under “Creating a User Function” on page 6-5.

Because the sensor-controlled User functions are a special type needing separate discussion. more information on them is provided under “Description of User Functions” on page A-12

Function Names Functions have abbreviated names which describe the action they perform. For example, Function 44, ACN to SynCol A, delivers acetonitrile to Column A. You can trace the pathway created by activating Function 44 on the simplified synthesizer module schematic shown in Figure A-1.

The third class of functions, Special Logical (Cycle Directive) functions, are functions whose behavior cannot be anticipated by relating the function name to the instrument's reagents or mechanical components.

Time and Misc FieldEntries or

Subroutines

In all cases, the Time and Misc fields are used as follows:

� The Time field uses a time in seconds and tenths of a second.

� The Misc (Miscellaneous) field is used to declare any other type of step parameter such as a pressure or temperature setting.

Sometimes a function's behavior is modified simply by having a non-zero instead of a zero in the Miscellaneous field. In these cases, any non-zero value will work. ABI cycles use 999 with these functions to distinguish between “non-zero” and the specific values required by other functions.


Tracing Flow Pathsfor Functions

To understand how the instrument performs the process of synthesis, compare the Synthesis cycle listing provided in Appendix B and the plumbing diagram on page A-9 with the following function lists:

� Table A-2 on page A-15


To understand the process of cleavage/deprotection, compare the Cleavage/Deprotection cycle listing provided in Appendix B and the appropriate portions of the synthesizer schematic in Appendix D with the following function lists:






To understand the process of purification/quantitation, compare the purification cycle listing provided in Appendix B and the appropriate portions of the synthesizer schematic in Appendix D with the following function lists:

Table A-8 on page A-18



To achieve reagent flow, specific valves must open simultaneously so that the following occurs:

� The pathway from the pressurized bottle to the valve block is opened.

� An exit is provided out of the valve block to waste, or to the column and then to either the waste port or the collection vial.


Figure A-1 Flow path of ACN to Column A (valves 1,15,22,26)(partial schematic of synthesis module only)

24

22

23

25

26

27

28

14

15

16

13

12

11

10

9

8

65

432

1

A

B

7

CH3CN

Tetrazole

Legend

= Valve

= Bottle

Synthesis Col A

Gas

(Reg 7B) Gas

CH3CN

Column ValveBlock D

Amidite 5

Reg. 4A30V

Amidite 6

Amidite 8Amidite 7

C

C

Synthesis Col B

Synthesis Col C

Amidite TAmidite CAmidite GAmidite A

To Block C

Spare

Halogenated Waste

Synthesis Col BSynthesis Col C

Flammable Waste

(Reg 7C)


Valve Functions

Types of ValveFunctions

There are three basic types of valve functions:

� Block functions - which do not deliver to columns or coils

� Serial functions - which deliver to a specific column or coil and will only execute if their designated column is active

� Parallel functions - which, within a given area such as cleavage, deliver to all active columns or coils at the same time

In an instance where only a single column is active, a parallel function will behave like the corresponding serial function dedicated to that one column.

Associated parallel and serial functions are always clustered together on the function list in the order: Parallel Function, Serial A, Serial B, Serial C.

Uses of ValveFunctions

Valve functions do the following:

� Direct the reagent deliveries in the Synthesis, Cleavage/Deprotection, and Purification cycles that are necessary to produce oligonucleotides on the 3948.

� Constitute the following procedures: bottle change, auto-dilution, begin, end, miscellaneous, and flow test.

These procedures as well as the three types of chemistry cycles are documented further on in this appendix.

A valve function works by opening or closing a valve or set of valves simultaneously to perform a specific delivery or task for a specified time, such as deliver reagents: Reagents delivered in this way flow through the column and then to either the waste bottle, deprotection coils, or the UV detector and the collection vial.

Manual Control Valve functions are used automatically by the system to perform chemistry or other tasks when you run a procedure or a chemistry protocol. If you desire, you can manually activate valves as well as other types of functions using the Manual Control view whenever the instrument is not active.

Creating Your OwnFunctions

Besides activating functions which are already defined, the ABI™ 3948 System provides you with the capability of creating your own functions. As described under “Exercising Manual Control of a Function” on page 6-4, you can create more than 100 user-defined functions in the Manual Control View and combine them with the standard set of functions to customize procedures.

Customized procedures may also be executed from within the Edit view used to create them (see Execute Button on page 6-14). During synthesis, the Monitor Chemistry menu displays each function as it is activated. For information on creating your own sensor-controlled functions, see “Description of User Functions” on page A-12.


Sensors

GeneralCharacteristics

Valve functions may be sensor controlled or not and work as follows:

Note With Flush and Back Flush functions, sensors are used to detect dryness. All other sensor functions execute until the sensor becomes wet.

Location of Sensors Sensors are located in these places:


Serial and Parallel compared to block

All serial and parallel functions work with liquid/gas sensors while block functions do not.

Execution by time or sensor

Sensor functions execute for the time specified in the step unless their sensor is activated (i.e., the step field “SNS” equals YES).

In this case, execution stops either when the sensor is triggered or when the time runs out, whichever occurs first (see the discussion under “Retries” below).

Parallel functions Parallel functions turn off the delivery to each column or coil separately as each different sensor triggers.

Location Description

Above jaw positions

On flow paths through the jaws that clamp on the OneStep™ columns, primarily above the columns to detect the deliveries of reagents through the columns.

The sensors above the columns are also used to verify that columns have been flushed dry with gas or that the cleavage or purification vessels have been drained (back flushed) until empty.

Below synthesis jaw positions

Between the reagent block and columns in the synthesis module to minimize the consumption of expensive amidites.

These sensors allow the delivery of short slugs of reagent that are then pushed into position to saturate the columns. The upper synthesis sensors control this final push so that it is stopped at the right time to leave these short slugs resting over the columns.

Near amidite bottles

On flow paths out of the primary amidite bottles (A, G, C, and T — bottles 1 through 4) to verify deliveries out of these bottles.

Near deprotection coils

On flow paths through the three deprotection coils to detect the arrival of samples into the coils.

Near the UV cell On the flow path through the UV cell to detect the arrival of a sample at the cell.


Retries Retries work as listed below:

Description of UserFunctions

The last 100 functions on the ABI 3948 System function list (SynUpr Wet 401 to 500 UV Dry 500) are user functions that can perform sensor-controlled deliveries. User functions have the following characteristics:

The particular sensor or set of sensors tied to a sensor user function is specified in the default function name. As with standard user functions, the function name may be edited as well as the valve list.

Components ofSensor User

Function Names

There are three components to the default names provided for sensor user functions:


Number of retries Any function executing with sensor(s) active will retry a delivery or dry up to six times if the sensor does not report success initially (except Base+Tet).

Any function that retries more than four times will issue a message to serve notice that trouble may be developing.

Note Ramping functions will retry only four times instead of the usual six and do not report retries, only failure

Execution until time elapses or trigger occurs

Each retry executes for as long as the time specified unless the sensor triggers during the retry and ends the function.

Fifteen seconds Delivery functions will wait fifteen seconds between retries to ensure bottle repressurizing between attempts.

Back flushes and drains

Drying functions (back flushes/drains and flushes) do not wait between retries.

Parallel functions Parallel functions will retry only those columns that have not already been successful and may retry each column a different number of times.

Characteristic Definition

Valve lists defined by operator

Like standard user functions, the valve list for these functions can be defined by the operator.

Sensors are pre-assigned

These functions offer the additional advantage of having sensors pre-assigned to them.

Engaged by YES in SNS field

Sensors are engaged in the usual fashion by setting the SNS field in the step to YES.

Components Description

1 The first part of the default name identifies the sensor or sensors associated with the function.

2 The second component spells out whether the function is ‘wet’ or ‘dry,’ that is, whether the function's sensor(s) trip upon detecting wetness (liquid delivery) or dryness (gas delivery, flush or back flush).

3 The final component of the default function name, in keeping with the convention for standard user functions, is the function number.


Names andLocations of Sensors

Sensors have the following names and locations:

See the Plumbing Diagram in Appendix D of this manual to verify which sensors are associated with which columns. In some instances, the column alphabetic order and sensor numeric order are reversed.

Use of Parallel withSerial Sensor User

Functions

As with other sensor functions, parallel sensor user functions are always grouped on the function list together with their associated serial functions. So SynUpr Wet 401 is followed by SynUprWet A 402, SynUprWet B 403, and SynUprWet C 404.

This grouping of sensor functions into sets is crucial to the operation of parallel user functions:

User definable functions operate according to the same scheme as follows.

� To program the valve list for a parallel user function, enter the appropriate valve lists for each associated serial function.

� To aid in documenting the valve assignments made for serial functions associated with a parallel user function, enter the complete three-column valve list for the serial functions into the listing for the parallel function.

The entire valve list may then be viewed under one function, the parallel function, in Manual Control.

Sensor Name/Number Location

SynUpr 1, 2, 3 Sensors with these designations are upper synthesis sensors.

SynMan 22, 23, 24 Sensors with these designations are synthesis manifold sensors.

SynLow 4, 5, 6 Sensors with these designations are lower synthesis sensors.

Clv 10, 11, 12 Sensors with these designations are cleavage sensors.

Coil 10, 11, 12 Sensors with these designations are coil sensors.

Pur 14, 15, 16 Sensors with these designations are purification sensors.

UV 21 This is the UV sensor.


Parallel use serial function valve lists

Parallel sensor-controlled functions do not use their own valve lists to execute, but use the valve lists of their associated serial functions instead.

Parallel turn on only valves for active columns

Parallel sensor-controlled functions turn on only the valves for active columns by merging together the valve list for each active column's associated serial function and excluding the valve lists of functions associated with inactive columns.

Valves for each column act independently

When one column finishes its delivery before the others, its single set of valves is deactivated. Any other active columns' valves will remain on until their sensors trigger.


Sensor-ControlledUser Function

Example

As explained above in the discussion of parallel user functions, sensor-controlled parallel user functions execute the valve list settings made in the associated serial functions. This means that sensor-controlled user functions are always used in groups of four functions, the parallel function for the group and the three serial functions associated with it.

For example, a user might want to deliver Tetrazole (TET) until it tripped the upper sensors (the sensors above the synthesis columns) instead of the manifold sensors as the standard TET to SynCols (Fxn 48) parallel function would do. This can be done using these four functions: SynUpr Wet 401, SynUpr Wet A 402, SynUpr Wet B 403, and SynUpr Wet 404.

Using these functions requires the following four steps:

The following valve list entries are required for the functions in this example:

Note The value set for trip time can be any value greater than actually needed for the sensor(s) to trip. The actual trip values will be reported to the Microphone file.

Step Action

1 Put the desired valve lists into the associated serial functions.

2 Open the cycle intended to execute the parallel function and turn the sensors on for the parallel function (“Yes” in the SNS field).

3 Set a trip time (value in seconds in the TIME field) greater than required for tripping sensors (perhaps 26 seconds).

4 Run the cycle in which you called the parallel function to execute it.

Function Valves

SynUpr Wet A 402 Valve 3 (Tetrazole)

Valve 15 (Column A)

SynUpr Wet B 403 Valve 3 (Tetrazole)

Valve 14 (Column B)

SynUpr Wet C 404 Valve 3 (Tetrazole)

Valve 13 (Column C)


Valve Functions by Functional Category

List of FunctionCategories

Valve functions are listed by functional category within this section. The third column of the function table for each category is used to indicate the type of Valve function (a blank, no entry, indicates a Serial function, Parallel indicates a Parallel Function, Ramping indicates a Ramping Function, and Block indicates a Block Function.

Note The functions in this category are used together with Non-valve Hardware functions and Logical or Cycle Directive functions to perform instrument chemistry.

To understand how valve functions work in a particular chemistry cycle, refer to the chemistry cycle listing in Appendix B and the material entitled “Special Functions” in this appendix to understand how the instrument performs chemistry. The Plumbing Diagram is provided in Appendix D to aid you in tracing out chemistry flow paths.

This section categorizes functions in the following areas:

� Synthesis Valve Functions

� Synthesis Column Flushing and Back Flushing Functions

� Cleavage Functions

� Purification Functions

� Bottle Change Functions

� User Functions

� Purification and Quantitation Functions

� Block Functions

Synthesis ValveFunctions

Table A-2 shows the Synthesis functions used for reagent deliveries to columns in synthesis jaws.

Table A-2 Synthesis Functions

Func.Number Function Name

Parallel/Block/Special Func.



1 B+ TET to Syns See “Special” functions.

17 8+ TET to Syn B

2 A+ TET to Syn A 18 A+ TET to SynC

3 G+ TET to Syn A 19 G+ TET to Syn C

4 C+ TET to Syn A 20 C+ TET to Syn C

5 T+ TET to Syn A 21 T+ TET to Syn C

6 5+ TET to Syn A 22 5+ TET to Syn C




10 A+ TET to Syn B 26 Coupling Wait See “Special” functions

11 G+ TET to Syn B 27 CAP to Syn Cols Parallel

12 C+ TET to Syn B 28 CAP to SynCol A

13 T+ TET to Syn B 29 CAP to SynCol B

14 5+ TET to Syn B 30 CAP to SynCol C


Synthesis valve functions provide for flow of reagents (driven by argon pressure) from the reservoirs, through valve blocks A, B, or C, through the OneStep columns, where all chemical reactions necessary for synthesis occur.

Two other types of functions, Flushing and Back Flushing, although they do not deliver reagents, are essential to Synthesis. These functions perform tasks such as flushing and back flushing columns (see Table A-3 below).

Synthesis ColumnFlushing and BackFlushing Functions

The functions in Table A-3 flush and back flush synthesis columns:

15 6+ TET to Syn B 33 IODINE to Syns Parallel

16 7+ TET to Syn B 34 IODINE to Syn A

37 IODINE to Waste Block 45 ACN to SynCol B

38 TCA to Syn Cols Parallel 46 ACN to SynCol C

39 TCA to SynCol A 47 ACN to SynWaste Block

40 TCA to SynCol B 48 TET to Syn Cols Parallel

41 TCA to SynCol C 49 TET to SynCol A

43 ACN to Syn Cols Parallel 50 TET to SynCol B

44 ACN to SynCol A 51 TET to SynCol C

Table A-2 Synthesis Functions (continued)





Table A-3 Synth-Gas Flushes and Back Flushes

FunctionNumber Function Name

Parallel/Block/Special Function

53 Flush Syn Cols Parallel

54 Flush Syn Col A

55 Flush Syn Col B

56 Flush Syn Col C

57 Back Flush Syns Parallel

58 Back Flush Syn A

59 Back Flush Syn B

60 Back Flush Syn C

61 Trityl Flush Syn Block

62 TritylFlsh SynA

63 TritylFlsh SynB

64 TritylFlsh SynC

65 SynLowBlk Flush Block


Cleavage Functions The following tasks are performed by functions listed in this section:

� Delivery to Clv Cols (Table A-4)

� Gas Flushes and Pressurization (Table A-5)

� Oligonucleotide Transfer (Table A-6)

� Water Deliver to Deprotection Coil (Table A-7)

Delivery to Cleavage Columns

The functions listed in Table A-4 prime delivery lines and deliver reagents to cleavage columns.

Gas Flushes and Pressurization

The functions listed in Table A-5 flush and back flush cleavage columns and deprotection coils:

Table A-4 Functions for Delivery of Reagents to Clv Cols





111 Ammonia to Clvs Parallel 126 H2O to Clv Cols Parallel

112 Ammonia to ClvA 127 H2O to ClvCol A

113 Ammonia to ClvB 128 H2O to ClvCol B

114 Ammonia to ClvC 129 H2O to ClvCol C

115 Ammonia toWaste Block 130 H2O to ClvWaste Block

116 ACN to Clv Cols Parallel

117 ACN to Clv A

118 ACN to Clv B

119 ACN to Clv C

120 ACN to ClvWaste Block

Table A-5 Functions for Gas Flushes and Pressurization





121 Gas to Clv Cols Parallel 134 Back Flsh Clv C

122 Gas to ClvCol A 138 Press Line Block

123 Gas to ClvCol B 144 Clv Blk Flush Block

124 Gas to ClvCol C 153 DepLowBlk Flush Block

125 Gas to Clv II Block 155 Gas toDep Coils Parallel

131 Back Flush Clvs Parallel 156 Gas to Coil A

132 Back Flsh Clv A 157 Gas to Coil B

133 Back Flsh Clv B 158 Gas to Coil C


Oligonucleotide Transfer

The functions listed in Table A-6 transfer oligonucleotides from the cleavage columns into the deprotection coils.

Water Delivery to Deprotection Coil

The functions listed in Table A-7 deliver water to the deprotection coils:

Purification andQuantitation

Functions

The following tasks are performed by functions listed in this section:

� Delivering Reagents to Purification Columns/UV Detector (Table A-8)

� Rinsing and Flushing Deprotection Columns (Table A-9)

� Collecting and Quantitating Samples (Table A-10)

Delivery of Reagents to Purification Columns/UV Detector

The functions listed in Table A-8 deliver reagents to the purification columns and the UV detector.

Table A-6 Functions for Oligonucleotide Transfer





141 Xfer Clv Col A 153 DepLowBlk Flush Block

142 Xfer Clv Col B 154 Dep Upr Blk Rinse

Block

143 Xfer Clv Col C 166 Xfr Coil A Ramping

151 Dep Xfer Rinse Block 167 Xfr Coil B Ramping

152 Dep Xfer Flush Block 168 Xfr Coil C Ramping

Table A-7 Functions to Deliver Water to Deprotection Coils


163 H2O to Coil A

164 H2O to Coil B

165 H2O to Coil C

Table A-8 Functions Delivering Reagents to Pur. Columns/UV Detector





173 ACN to Pur Cols Parallel 190 TFA to PurCol B

174 ACN to Pur ColA 191 TFA to PurCol C

175 ACN to Pur ColB 193 20%ACN to Purs Parallel

176 ACN to Pur ColC 194 20%ACN to Pur A

177 ACN to PurWaste Block 195 20%ACN to Pur B

178 TEAA to PurCols Parallel 196 20%ACN to Pur C

179 TEAA to Pur A 197 20%ACN to Waste

180 TEAA to Pur B 198 AcAcid to Purs Parallel

181 TEAA to Pur C 199 AcAcid to Pur A

182 TEAA to Waste Block 200 AcAcid to Pur B


Rinsing and Flushing Deprotection Columns

The functions in Table A-9 rinse and flush the deprotection columns:

Collecting and Quantitating Samples

The functions listed in Table A-10 collect and quantitate samples:

183 H2O to Pur Cols Parallel 201 AcAcid to Pur C

184 H2O to PurCol A 202 AcAcid to Waste Block

185 H2O to PurCol B 211 Pur A to UV

186 H2O to PurCol C 212 Pur B to UV

188 TFA to Pur Cols Parallel 213 Pur C to UV

189 TFA to PurCol A 214 H2O to UV Block

Table A-9 Functions for Rinsing and Flushing Deprotection Columns





171 Pur Xfer Rinse Block 208 Back Flsh Pur A

172 Pur Xfer Flush Block 209 Back Flsh Pur B

203 Gas to Pur Cols Parallel 210 Back Flsh Pur C

204 Gas to PurCol A 218 PurLowBlk Flush Block

205 Gas to PurCol B 219 PurUprBlk Flush Block

206 Gas to PurCol C 220 Pur Vent Block

207 Back Flush Purs Parallel 222 SC Block Flush Block

Table A-10 Functions for Collecting and Quantitating Samples



135 Crude A to UV Ramping

136 Crude B to UV Ramping

137 Crude C to UV Ramping

211 Pur A to UV Ramping

212 Pur B to UV Ramping

213 Pur C to UV Ramping

215 Xfer UV to SC Block

217 Flsh UV toWaste Block

Table A-8 Functions Delivering Reagents to Pur. Columns/UV Detector (continued)






Bottle ChangeFunctions

The functions listed in Table A-11 are used during bottle change procedures:

User Functions User Functions are a reserved class of valve function which enables the user to define functions using any valve listed in the Manual Control View. The empty functions listed in Table A-12 are available for user definition:

Note The functions in the range 401–500 are discussed under “Description of User Functions” on page A-12.

Table A-11 Bottle Change Functions



82 ACN AMIDITE A 95 Mix AMIDITE 5

83 ACN AMIDITE G 96 Mix AMIDITE 6

84 ACN AMIDITE C 97 Mix AMIDITE 7

85 ACN AMIDITE T 98 Mix AMIDITE 8

86 ACN AMIDITE 5 87 Mix AG

87 ACN AMIDITE 6 98 Mix AGCT

88 ACN AMIDITE 7 99 Mix AGCT

89 ACN AMIDITE 8 100 Mix AG

90 Mix AMIDITE A 101 Mix CT

91 Mix AMIDITE G 102 AMIDITE Vent

92 Mix AMIDITE C 103 Ammonia Vent

93 Mix AMIDITE T 104 Mix Ammonia

Table A-12 Functions for User Definition (User Functions)

150 383 389 380 386 392

227 384 390 381 387 393

228 385 391 382 388 401-500


Block Functions Block functions are functions which do not deliver to column or deprotection coils and include functions which perform the following tasks:

� Priming Delivery Lines - below

� Priming Functions for Cleavage Columns - below

� Priming Functions for Purification Columns - on page A-22

� Gas Flush, Pressurization, and Rinse - on page A-22

� Venting Functions for Block and Reagent Bottles - on page A-22

Priming Delivery Lines

The functions in Table A-13 are those used to prime delivery lines.

Priming Functions for Cleavage Columns

The functions in Table A-14 prime cleavage columns:

Table A-13 Functions for Priming Delivery Lines


Function Number Function Name

31 AC2O to Waste 115 Ammonia toWaste

32 NMI to Waste 120 ACN to ClvWaste

37 IODINE to Waste

42 TCA to Trityl

47 ACN to SynWaste

52 TET to Waste

74 A AMIDITE Waste

75 G AMIDITE Waste

76 C AMIDITE Waste

77 T AMIDITE Waste

78 5 AMIDITE Waste

79 6 AMIDITE Waste

80 7 AMIDITE Waste

81 8 AMIDITE Waste

Table A-14 Functions for Priming Clv Cols


115 Ammonia to Waste

120 ACN to ClvWaste

130 H2O to ClvWaste


Priming Functions for Purification Columns

The functions in Table A-15 prime purification columns:

Gas Flush, Pressurization, and Rinse Functions

The functions in Table A-16 are used to flush, back flush, pressurize valve blocks and various system components, and perform block rinses:

Venting Functions for Block and Reagent Bottles

The functions in Table A-17 are used to vent various blocks and reagent bottles:

Table A-15 Functions for Priming Purification Columns

177 ACN to PurWaste

182 TEAA to Waste

187 H2O to PurWaste

192 TFA to Waste

197 20%ACN to Waste

202 ACAcid to Waste

Table A-16 Functions for Gas Flushes, Pressurization, and Rinses


Function Number Function Name

65 SynLowBlk Flush 132 Back Flush Clv A

105 SynUprBlk Flush 133 Back Flush Clv B

106 LowTrityl Flush 134 Back Flush Clv C

107 UprTrityl Flush 138 Press Line

122 Gas to ClvColA 139 Compress

123 Gas to ClvColB 140 Clv Vent

124 Gas to ClvColC 151 Dep Xfer Rinse

125 Gas to Clv II 144 Clv Blk Flush

132 Back Flush Clv A 152 Dep Xfer Flush

133 Back Flush Clv B 153 DepLowBlk Flush

134 Back Flush Clv C 154 DepUprBlk Rinse

138 Press Line 171 Pur Xfer Rinse

139 Compress 172 Pur Xfer Flush

140 Clv Vent 218 PurLowBlk Flush

151 Dep Xfer Rinse 219 PurUprBlk Flush

144 Clv Blk Flush 221 DepUprBlk Flush

152 Dep Xfer Flush 222 SC Block Flush

Table A-17 Functions for Block Venting



66 Syn Upper Vent 101 AMIDITE Vent

67 Syn Lower Vent 102 Ammonia Vent


Non-Valve Hardware Functions

Overview Non-valve hardware functions encompass all hardware related functions except for valves and include the following:

� Pressure Regulating Functions

� Calibration Functions

� Turntable Control Functions

� Miscellaneous Functions (UV, Relay, Heater/Fan, Toggle)

� Jaw Functions

� Sample Collector Functions

Pressure RegulatingFunctions

The functions in Table A-18 regulate system pressures:

CalibrationFunctions

The functions in Table A-19 calibrate system pressure as well as gas and liquid sensors:

Turntable ControlFunctions

The functions in Table A-20 control the turntable:

Table A-18 Functions to Regulate Pressure



301 Set Pres Reg 1 309 Set Pres Reg 9

302 Set Pres Reg 2 310 Set Pres Reg 10

303 Set Pres Reg 3 311 Set Press RegAll

304 Set Pres Reg 4 312 Press Reg On

305 Set Pres Reg 5 313 Press Reg Off

306 Set Pres Reg 6 314 Pres Reg AllOn

307 Set Pres Reg 7 315 Pres Reg All Off

Table A-19 Functions to Calibrate Pressure and Sensors



320 Check Snsr Cal 323 Calib AllGasSns

321 CalibrateGasSns 324 Calib AllLiqSns

322 CalibrateLiqSns

Table A-20 Turntable Control Functions


239 TurnTable Home

240 TurnTable Next

241 TurnTable Prev


MiscellaneousHardware Functions

The functions in Table A-21 control relays, fan, heater, Set Temperature, and Toggle Valve functions:

Jaw Functions The functions in Table A-22 control the jaws in the three chemistry modules:

Sample CollectorFunctions

The functions in Table A-23 control the sample collector:

Table A-21 Hardware Control Functions



216 UV Reading 170 Start Depro Htr

223 Set UV Scale 255 Fan On

249 Relay 1 On 256 Fan Off

250 Relay 1 Off 257 Heater On

251 Relay 1 Pulse 258 Heater Off

252 Relay 2 On 260 Set Coil Temp

253 Relay 2 Off 261 Wait Coil Temp

254 Relay 2 Pulse 262 Tggle Vlves On

169 Depro Htr Wait 263 Tggle Vlves Off

Table A-22 Jaw Functions


231 Syn Jaw Open

232 Syn Jaw Close

233 Clv Jaw Open

234 Clv Jaw Close

235 Pur Jaw Open

236 Pur Jaw Close

237 Open All Jaws

238 Close All Jaws

Table A-23 Sample Collector Functions


242 SC Waste

243 SC Home

243 SC Next

244 SC Open

245 SC Close

246 SC Needle Up

247 SC Needle Down

264 SC Prev

ABI 3948 System Special Functions B-1

ABI 3948 System Special Functions B

In This Appendix

Topics Covered This appendix contains a detailed listing of ABI™ 3948 System Special Functions.

The appendix contains the following topics:

Topic See page

Overview of ABI 3948 System Special Functions B-2

Introduction B-2

Listing of Special Functions B-5

B

B-2 ABI 3948 System Special Functions

Overview of Special Functions

Introduction A number of “special” functions have been developed for the ABI 3948 System. These are functions whose behavior cannot be anticipated by relating the function name to the instrument's reagents or mechanical components.

Often, these functions are normal sensor functions that have certain extra capabilities built into them. Typically, these extra features are engaged by setting values in the “MISC” parameter field of a step. All of the logical or cycle directive functions fall into this class of special functions and usually invoke the Miscellaneous field as well.

The special functions listed in Table B-1 are discussed in order of function number in the next subsection. Where a group of functions are all of the same type or work in conjunction with each other, they are discussed together at the point where the first function of the group appears on the list.

For ready reference, functions are always listed once according to their position on the function list. This ordered listing will contain a reference to an earlier listing if the function was discussed previously as part of a function group.

Note Function 110 will report the database version number found in your instrument.

Table B-1 Table of Special Functions




1 B+ TET to Syns 176 ACN to PurCol C 230 Go Sub On Fail

26 Coupling Wait 179 TEAA to PurCol A 232 Syn Jaw Close

39 TCA to Syn Col A 180 TEAA to PurCol B 234 Clv Jaw Close

40 TCA to Syn Col B 181 TEAA to PurCol C 236 Pur Jaw Close

41 TCA to Syn Col C 184 H2O to PurCol A 251 Relay 1 Pulse

44 ACN to SynCol A 185 H2O to PurCol B 254 Relay 2 Pulse

45 ACN to SynCol B 186 H2O to PurCol C 259 Wait

46 ACN to SynCol C 189 TFA to PurCol A 260 Set Coil Temp

109 Waste to Trityl 190 TFA to PurCol B 261 Wait Coil Temp

110 Database v4.20 191 TFA to PurCol C 262 Tggle Vlves On

135 Crude A to UV 194 20%ACN to Pur A 263 Tggle Vlves Off

136 Crude B to UV 195 20%ACN to Pur B 265 Set Rmp Fxn Sns

137 Crude C to UV 196 20%ACN to Pur C 266 Set Rmp Fxn Trp

141 Xfer Clv Col A 199 AcAcid to Pur A 267 Set Rmp Fxn Chk

142 Xfer Clv Col B 200 AcAcid to Pur B 268 Set Rmp Fxn Dly

143 Xfer Clv Col C 201 AcAcid to Pur C 269 Send Message

166 Xfer Coil A 207 Back Flush Purs 270 Comment

167 Xfer Coil B 211 Pur A to UV 271 Begin of Cycle

168 Xfer Coil C 212 Pur B to UV 272 End of Cycle

169 Depro Htr Wait 213 Pur C to UV 273 Begin Loop

170 Start Depro Htr 216 UV Reading 274 End Loop

174 ACN to PurCol A 223 Set UV Scale 275 Start Here

175 ACN to PurCol B 229 End Row SCP/123 276 Exit DMT On


277 If Pur Col A 407 SynUprWet B 407 447 Clv Dry B 447

278 If Pur Col B 408 SynUprWet C 408 448 Clv Dry C 448

279 If Pur Col C 409 SynUpr Wet 409 449 Clv Dry449

280 Else 410 SynUprWet A 410 450 Clv Dry A 450

281 Endif 411 SynUprWet B 411 451 Clv Dry B 451

282 Select Pur Cols 412 SynUprWet C 412 452 Clv Dry C 452

283 Select Act Cols 413 SynUpr Dry 413 453 Coil Wet 453

284 Clv Wants Depro 414 SynUprDry A 414 454 Coil Wet A 454

285 Pur Owns Depro 415 SynUprDry B 415 455 Coil Wet B 455

286 Clv Owns Depro 416 SynUprDry C 416 456 Coil Wet C 456

287 Go Sub 417 SynMan Wet 417 457 Coil Dry 457

288 Return Sub 418 SynManWet A 418 458 Coil Dry A 458

289 Sub Label 419 SynManWet B 419 459 Coil Dry B 459

290 Goto End 420 SynManWet C 420 460 Coil Dry C 460

291 If Any Act Cols 421 SynMan Wet 421 461 Pur Wet 461

292 Go Sub A 422 SynManWet A 422 462 Pur Wet 462

293 Go Sub B 423 SynManWet B 423 463 Pur Wet 463

294 Go Sub C 424 SynManWet C 424 464 Pur Wet 464

295 If First Cycle 425 SynMan Wet 425 465 Pur Wet 465

296 If Last Cycle 426 SynManWet A 426 466 Pur Wet 466

297 If Crude Col A 427 SynManWet B 427 467 Pur Wet 467

298 If Crude Col B 428 SynManWet C 428 468 Pur Wet 468

299 If Crude Col C 429 SynLow Wet 429 469 Pur Wet 469

300 Engage All Cols 430 SynLowWet A 430 470 Pur Wet A 470

316 If Cyc Greater 431 SynLowWet B 431 471 Pur Wet B 471

317 Save Reg Pres 432 SynLowWet C 432 472 Pur Wet C 472

318 Restore RegPres 433 SynLow Dry 433 473 Pur Wet 473

319 Revive Act Cols 434 SynLowDry A 434 474 Pur Wet A 474

320 Check Snsr Cal 435 SynLowDry B 435 475 Pur Wet B 475

325 Jaw Test Times 436 SynLowDry C 436 476 Pur Wet C 476

329 Begin Leak Test 437 Clv Wet 437 476 Pur Wet 477

330 End Leak Test 438 Clv Wet A 438 478 Pur Wet 478

396 Time Stamp 439 Clv Wet B 439 479 Pur Wet 479

400 * Reserved * 440 Clv Wet C 440 480 Pur Wet 480

401 SynUpr Wet 401 441 Clv Wet 441 481 Pur Dry 481

402 SynUprWet A 402 442 Clv Wet A 442 482 Pur Dry A 482

403 SynUprWet B 403 443 Clv Wet B 443 483 Pur Dry B y 483

404 SynUprWet C 404 444 Clv Wet C 444 484 Pur Dry C 484

405 SynUpr Wet 405 445 Clv Dry 445 485 Pur Dry 485

406 SynUprWet A 4 06 446 Clv Dry A 446 486 Pur Dry A 486

Table B-1 Table of Special Functions (continued)





487 Pur Dry B 487 492 UV Wet 492 497 UV Wet 497

488 Pur Dry C 488 493 UV Wet 493 498 UV Dry 498

489 UV Wet 489 494 UV Wet 494 499 UV Dry 499

490 UV Wet 490 495 UV Wet 495 500 UV Dry 500

491 UV Wet 491 496 UV Wet 496

Table B-1 Table of Special Functions (continued)





Listing of Special Functions

This subsection provides a discussion for each of the ABI™ special functions listed in Table B-1 in the previous subsection.

Functions are listed in two ways:

� Individual functions are listed by number and name to the left, as is done for “1 B+Tet to Syns” below.

In some cases, the only material provided for an individual function will be a reference to where the function is discussed as part of a group of functions.

� Functions discussed as a group will be listed at the point in the list where the first function of the group appears on the list. In these cases, all the functions in the group are listed in a table before the discussion.

1 B+TET to Syns

This function appears to be a parallel function but in actuality it is three serial functions combined as each base must be added to each column individually. When viewed in Manual Control, the valve list shown is very short because the complete valve list for this function is largely determined by two things: 1) the base being added to a particular column and 2) which columns are currently active.

This is the only sensor function that does not have the capacity to auto-resume (see function 232, Syn Jaw Close, et al.). This is also the only function that may check two sensors to verify delivery to a single column: a given column's synthesis manifold sensor is used to terminate delivery while base delivery is verified by sensors on some amidite bottles (bottles 1 to 4 have sensors and bottles 5 to 8 do not).

For each column that is active, B+ TET to Syns execution goes through two stages: delivering the amidite combined with tetrazole and then rinsing out the block with tetrazole alone. With all columns active, this function must execute both stages three times over. The B+TET Calibration Table specifies the delivery times for each stage given any combination of bottle being delivered from and column being delivered to. The B+TET times in this table are the maximum times allowed for the delivery sensors to trip. The TET to Col times are fixed delivery times and vary with the number of valve ports between the bottle being accessed and the column being delivered to.

26 Coupling Wait This function acts as a wait step. The time for the wait does not come from the time field, however, and any time listed in the step is ignored. The wait time is determined by the Base + TET Calibration Table. If the various bases in the current cycle have different coupling wait times, the longest time in the table for this set of amidites is the wait time used by this function.

39 TCA to SynCol A

The discussion below the table applies to all the functions listed in the table.

39 TCA to SynCol A to 41 TCA to SynCol C

44 ACN to SynCol A to 46 ACN to SynCol C

174 ACN to PurCol A to 176 ACN to PurCol C

179 TEAA to PurCol A to 181 TEAA to PurCol C

184 H2O to PurCols A to 186 H2O to PurCols C

189 TFA to PurCols A to 191 TFA to PurCols C


Whenever their associated column is inactive and the Miscellaneous (MISC) field of the step is non-zero, the functions listed above will wait instead of executing for the allotted step time (TIME). This is useful when performing push/wait loops where pushes are done serially and the wait time for one column is defined in whole or in part by the time taken to push reagents to the other columns.

109 Waste to Trityl This function is used to temporarily direct waste leaving the upper synthesis block to the trityl (halogenated) waste bottle when executing functions that would normally deliver into the flammable waste bottle. This allows certain housekeeping steps in synthesis to be used with both halogenated and non-halogenated reagents.

When Waste to Trityl is executed with a non-zero value in the Miscellaneous (MISC) field, it sets up the valve controller to substitute valve 23 for valve 22 whenever valve 22 is called for by a function. Execution will not revert to normal until this function is called with a zero in the Miscellaneous field.

110 Database Version

This is a function reserved to have its name show the version number of the database being used by the ABI 3948 System.

135 Crude A to UV The discussion below the table applies to all the functions listed in the table.

Collectively, the functions listed above are known as ramping functions. When executing with sensors active and a regulator number listed in the Miscellaneous (MISC) field, these functions will increase (ramp) the pressure on the specified regulator by one psi on each retry. Ramping functions will retry only four times instead of the usual six and do not report retries, only failures. When a ramping function sensor triggers, the function waits momentarily and then rechecks the sensor to verify the continued presence of liquid. The behavior of these functions is defined in part by the Set Rmp Fxn functions described later (functions 265–268), which functions must be called before these ramping functions can be used.

The Xfer Clv Col functions are a special case of ramping function. If these functions have not been successful by the conclusion of the final ramp, a recovery will be attempted. When a recovery is required, it is usually the result of a leaking jaw. Such a leak can prevent the ammonia slug containing the cleaved oligo from being effectively pushed toward the coils after it has passed through the jaws. (While the liquid slug is moving through the jaws, it helps to hold a seal. Once it passes, gas can leak rapidly and bleed off the pressure needed to keep the slug moving.)

The recovery deactivates the valve that delivers from the relevant cleavage vessel into the lower cleavage block (valve 33, 34, or 35) and attempts a final retry with the lower cleavage block pressurization valve activated instead (valve 29). The effect of this recovery is to flush into the coils any cleaved oligo that might be caught in the transfer

194 20%ACN to Pur A to 196 20%ACN to Pur C

199 AcAcid to Pur A to 201 AcAcid to Pur C

135 Crude A to UV to 137 Crude C to UV

141 Xfer Clv Col A to 143 Xfer Clv Col C

166 Xfer Coil A to 168 Xfer Coil C

211 Pur A to UV to 213 Pur C to UV


path from the lower cleavage block to the deprotection system. Before this recovery flush begins, the pressure is first reduced by two psi.

169 Depro Htr Wait

For this function, the Miscellaneous (MISC) field is used to declare the minimum number of minutes the deprotection heater must be on in order for deprotection to be completed. The Miscellaneous field is used to declare time in whole minutes since the maximum allowable entry in seconds (480) is not long enough to assure complete deprotection. If the heater has not been on long enough, the function calculates the remaining time it must wait and then waits for that period. If the cycle step calls for a zero wait time, this function will substitute the default wait time specified by the “Deprotect Mins” setup variable in the Instrument Preferences Window. This function is used to guarantee complete deprotection despite any variability in cycles or instrument performance.

170 Start Depro Htr

This function is essentially identical to function 260, Set Coil Temp, except that it informs the 3948 that the heater has been turned on specifically to begin deprotection. This allows the clock for Depro Htr Wait (above) to begin counting time once the heater has become hot. The temperature setpoint is entered in degrees C in the Miscellaneous (MISC) field. If the cycle step calls for a zero deprotection temperature, this function will substitute the default temperature specified by the “Deprotect Temp” setup variable in the Instrument Preferences Window.

174 ACN to PurCol A


See preceding discussion of function 39,TCA to Syn Col A, et al.

174 ACN to PurCol A to 176 ACN to PurCol C

179 TEAA to PurCol A to 181 TEAA to PurCol C

184 H2O to PurCol A to 186 H2O to PurCol C

189 TFA to PurCol A to 191 TFA to PurCol C

194 20%ACN to Pur A to 196 20%ACN to Pur C

199 AcAcid to Pur A to 201 AcAcid to Pur C


207 Back Flush Purs

This function is a normal sensor function that is able to ramp regulator pressures on retries. As usual for a sensor function, it will retry up to six times. In addition, if a regulator number is specified in the Miscellaneous (MISC) field, this function will ramp that regulator one psi on each retry. Unlike a true ramping function, it does not verify a sensor trigger and the function behavior is not affected by the ramping functions (see function 135, Crude to UV, et al.).

All Flush or Back Flush functions have the ramping capability. Since they are executed with the initial pressure always set to the maximum, there is no point in specifying a pressure ramp. Back Flush Purs, however, is used at a low initial pressure—but only in one instance. This occurs during purification when a slow drain of the vessel is used in order to best bind the deprotected oligo to the column. Ramping is specified in this instance to ensure a complete emptying of the vessel(s) when binding.

211 Pur A to UVto

213 Pur C to UV

See preceding discussion of function 135, Crude A to UV, et al.

216 UV Reading A value of zero in the Miscellaneous (MISC) field instructs UV Reading to take a baseline reading. This is generally done when the UV cell is filled with water. A value of 1, 2, or 3 calls for the taking of an oligo quantitation reading for the A, B, or C column, respectively. The most recent baseline reading is subtracted from the quantitation reading and the resulting o.d.u. and pmol/mL values are logged for reporting to the Run File at the end of the run.

223 Set UV Scale This function can be used to scale the translation of UV voltage readings into ODU. The Miscellaneous (MISC) field is used to define the scaling factor in percent. The scaling factor defaults to 100 without the intervention of this function. A scaling factor less than 100 will produce lower ODU readings and a factor greater than 100 will amplify the ODU values reported. Any change to this scaling factor will persist until changed again.

229 End Row SCP/ 123

End Row SCP/123 allows the operator to terminate chemistry on a single row of oligos without ending the entire run. The Miscellaneous (MISC) field is used to specify which row is to be terminated: 1 for synthesis, 2 for cleavage, and 3 for purification (SCP/123). This function operates only in Manual Control so a run must be Interrupted before it can be used. Upon resumption of the run, any cycle containing a terminated row will end immediately.

Some care must be exercised when attempting to end a row during a Pause After interrupt and, in general, it is preferable to avoid doing this altogether. At this point, synthesis and cleavage both contain the same row to be ended but for the End Row to take effect, the row would have to be ended in cleavage. This is because the synthesis row data has been copied over to the cleavage cycle but, in order to fully implement run extension, the synthesis cycle is not updated with the information on its new row until the run is resumed.

As an example, if a run is paused after synthesizing row 3, ending the row in purification would terminate row 2 while ending the row in cleavage would cancel row 3. Attempting at this point to end row 3 in synthesis would not be successful because control of the un-ended row has already been handed over to cleavage. In this scenario, to end row 4 without beginning chemistry on it, the run must be resumed


and an operator Interrupt set while the jaws are closing and pressure testing. Once this Interrupt has taken effect, row 4 may be ended using End Row SCP/123. So while it is tricky to correctly end a row at a Pause After, the Pause After feature may be used to enable the operator to end a row after the first step of the next cycle (jaw close).

There are several scenarios where End Row SCP/123 could prove useful. If, for example, synthesis falters on the final row because of an amidite shortage then the row can be terminated while the oligos in cleavage and purification continue on to conclusion. Or a row with a leaky OneStep™ column that appears in the middle of a run can be skipped over while the other oligos, both before and after that row, are run to completion.

230 Go Sub On Fail

Go Sub On Fail provides a means to interject a clean-up or recovery process into a cycle at the point where a critical delivery has failed. This function recognizes that a sensor delivery pause is pending and conditionally executes the subroutine number specified in the Miscellaneous field. (For more on subroutines, see function 287, Go Sub, et al.)

One key aspect of using this function is that the Go Sub On Fail step and all subsequent steps in the recovery subroutine must be flagged as Unsafe so that the run is not paused prior to the recovery being completed.

With any sensor delivery failure, the run resumes at the step that failed, regardless of the number of steps between the failure and the time the pause takes effect. Possible uses of a Go Sub On Fail subroutine include preparing a row for a graceful cancellation (see function 229, End Row SCP/123) or properly resetting conditions for a successful retry of the failed delivery.

232 Syn Jaw Close The discussion below the table applies to all the functions listed in the table

The Jaw Close functions listed above can be used without either a Time or a Miscellaneous (MISC) parameter to simply close the jaws. They may also use these parameters to optionally invoke a pressure test of the jaw seal when the jaw closes. In this case, the pressure drop test time is defined by the TIME field. The maximum allowable pressure drop is declared in the Miscellaneous field in 1/1000's of a psi.

If the “Pause On Jaw Leak” check box in the Instrument Preferences Window is checked and the jaw test fails, the run will be paused at that point. This may be the preferred option when the instrument is running with an operator in attendance. If the “End Row On Jaw Leak” check box in the Instrument Preferences Window is checked and the jaw test fails, all columns in the module associated with the failed jaw will be deactivated immediately (see function 229, End Row SCP/123). For throughput reasons, this is a useful option for an unattended instrument.

To perform a jaw/block pressure test, declare a non-zero pass/fail value for the pressure drop in the Miscellaneous field. If no pass/fail value is declared in the step, the default specified by the “Leak OK in 0.01 PSI” setup variable in the Instrument Preferences Window will be substituted. If the step and default values are both zero,

232 Syn Jaw Close

234 Clv Jaw Close

236 Pur Jaw Close

325 Jaw Test Times

Auto-resume


no jaw/block pressure test will be performed. The “Man Cont Jaw Testing” check box in the Instrument Preferences window must be checked to enable jaw/block pressure testing based on default values when closing the jaws in manual control. Jaw testing can also be initiated in manual control by specifying the test parameters when executing the jaw close.

Jaw Test Times uses the TIME field to define the default pressure drop time for any jaw/block pressure test. The initial pressurization time in whole seconds (settling time) is defined in the Miscellaneous field. If this function is not used to define these times, a default of 30 seconds will be used for both. The times established by this function must be reinstated after a power failure or database reset.

The Instrument Preferences window contains the testing pressure in psi for jaw/block pressure tests performed within cycles and defines the terms under which the auto-resume feature will be enabled. The term “auto-resume” refers to the 3948's ability to wait for a period and then restart itself after being paused by a sensor delivery failure. The auto-resume feature is closely tied to the results of the jaw/block pressure tests and so it is discussed here.

For auto-resume to be enabled, three basic requirements must be met. The first requirement is for the operator to select the correct settings in the Instrument Preferences Window. The default settings accomplish this. Secondly, the system must be shown to be currently leak tight so that auto-resuming will not result in magnifying spillage due to a leak. Depending on the results of each individual jaw/block leak test, some active cycles may not be allowed to auto-resume at the same time that others would be. Finally, auto-resume is an automatic process designed for unattended operation: control is always surrendered to the operator if there is any manual intervention with the instrument while an auto-resume is pending.

To meet all three conditions, a number of factors must be in place:

� “Pause On Sensor Fail” must be checked in the Instrument Preferences Window so that sensor delivery pausing is enabled.

� “Auto-resume Minutes” in the Instrument Preferences Window must be set to some value greater than zero (15 minutes is the default).

� Jaw/block pressure testing must be in effect and the testing pressure must be done at the system maximum 12 psi as specified by the “Jaw Leak Test in PSI” setup variable in the Instrument Preferences Window. A value of 12 psi is the default for this setup variable.

� Jaw/block pressure testing must be performed with a minimum pressure drop time of 30 seconds. This is the default time provided for by the system but care must be used if this time is modified either within the jaw close step or by setting an alternate default using Jaw Test Times.

� Jaw/block testing must past the “Auto-res OK 0.01 PSI” test standard in the Instrument Preferences Window. This allows a maximum pressure drop of only 1.80 psi to pass compared to a maximum allowable drop of 5 psi for the “Leak OK in 0.01 PSI” preference value.

These setup variable default values are 1.30 and 2.00 psi, respectively (entered in 1/100's p.s.i. as 130 and 200). It is possible for the instrument to “pass” the jaw/block leak test and run chemistry while not passing a more stringent standard required to enable auto-resume.

� The instrument must be running automatically. Any intervention by an operator during an auto-resume wait period will cancel the auto-resume. Such operator


interventions include initiating a manual control action, manually resuming the run, or even “interrupting” an instrument that is paused with an auto-resume pending. Also, auto-resume will never go into effect while a procedure or other operation is underway in manual control. However, once a run is resumed (by the operator), auto-resuming is re-enabled and will go into effect if there are any future sensor delivery failures within the cycle.

If the above conditions are met, most sensor delivery functions will auto-resume when they fail to deliver. The exceptions to this are function number 1, B+TET to Syns, and ramping functions (see the preceding discussion of function 135, Crude A to UV, et al).

251 Relay 1 Pulseand

254 Relay 2 Pulse

Different external devices, such as fraction collectors, may require different length pulses to be controlled properly. The pulse width for these functions is determined by the time entered for the step. Additionally, relay 2 is turned on when a run is Interrupted, is turned off when the run is Resumed, and is pulsed for two-tenths of a second when a run ends. This is done to allow relay 2 to trigger an alarm or other device to alert the operator when a run has paused or ended.

260 Set Coil Temp This function sets the deprotection heater to the target temperature specified in the Miscellaneous (MISC) field (in degrees C). If the Miscellaneous field is zero, this function will substitute the default value specified by the “Xfer Into Coil” setup variable in the Instrument Preferences Window.

261 Wait Coil Temp

This function sets the deprotection heater to the target temperature specified in the Miscellaneous (MISC) field (in degrees C). It then waits the allotted Time for the specified temperature to be reached. Like a sensor function, if the temperature is reached before time runs out, the function ends early. If the step runs out of time and the temperature is further than 2 °C from its target, a message is issued. If the cycle step calls for either a zero temperature or a zero Time (or both), this function will substitute the appropriate default value(s) specified in the Instrument Preferences window (the “Coil Cool Secs” and “Xfer From Coil” setup variables.)

262 Tggle Vlves Onand

263 Tggle VlvesOff

Toggling on a valve is like turning on a light in a room. No matter what else happens in the room, the light remains on until it is turned off. Tggle Vlves On turns on the valve specified in the Miscellaneous (MISC) field so that it stays on throughout the execution of other functions. Tggle Vlves Off is used to turn the valve off again. A set of valves must be toggled on and off one at a time. This feature is used while draining TFA during purification. At that time, Valve 70 is then toggled so that it temporarily redirects vessel drains into the halogenated waste bottle.

265 Set Rmp FxnSns


The Set Rmp Fxn functions listed above are used to set parameters that help to define the behavior of the ramping functions described previously (see function 135,Crude to UV, et al.). These parameters must be set in each chemistry controller before ramping functions can be successfully used by that controller. There are four chemistry

265 Set Rmp Fxn Sns

266 Set Rmp Fxn Trp

267 Set Rmp Fxn Chk

268 Set Rmp Fxn Dly


controllers: synthesis, cleavage, purification, and manual control. The parameter values may be set differently for each controller. The parameter values are declared in the Miscellaneous (MISC) field of a Set Rmp Fxn function and set by executing the function within a given chemistry controller (i.e., within a cycle). Once set, these parameters will persist until changed.

When activated, sensors continuously sample for the presence of wetness every 12 milliseconds and record a series of “wet” and “dry” readings. Each sensor decides whether the flow path is wet or dry based on some number of the most recent readings taken and the proportion of those readings that is wet or dry. Except for ramping functions, these standards for wet/dry are fixed. The Set Rmp Fxn's allow the cycle developer to vary these values for ramping functions.

Set Rmp Fxn Sns defines how large a sampling of most recent readings will be evaluated to decide if the sensor flow path is wet or dry. This number cannot be larger than 10, the value that is assigned to most non-ramping functions.

Set Rmp Fxn Trp defines how many of the sample readings must be “wet” to trigger—or trip—the sensor. When dealing with cool, non-gaseous liquids it is reasonable to set this value high relative to the number of samples assessed (e.g., 9 “wets” out of a set of 10 readings). When dealing with hot, frothy ammonia coming out of the deprotection coils, a lower standard such as 5 “wets” out of 10 readings is reasonable.

The “Sns” and “Trp” values are built into all sensor functions but the ramping functions additionally verify a liquid slug once detected. Set Rmp Fxn Chk defines the number of “wets” required to verify—or check—the initial trigger and end function execution if verified. For a 9 out of 10 wet trigger, a 7 out of 10 verify is reasonable and a 5 out of 10 verify is fine for hot ammonia.

Set Rmp Fxn Dly defines the number of milliseconds to pause (delay) after the initial trigger before beginning to collect readings for the verification. To avoid losing too much sample past the sensor during verification, this delay should not be too long—50 milliseconds is typical.

If a ramping function does not verify within two and one-half seconds, it resets the sensor to look again for a new trip followed by a verify. Verification is especially useful during critical transfers because it allows the function to overlook any small slugs that might precede the principal slug. Without verification, the actual sample could be lost whenever a leading slug tricked a transfer into stopping too early.

It is technically possible for a ramping function to trip many times before verifying. In practice, a second trip is infrequent and a third trip has never been observed.


269 Send Messageand

270 Comment

Send Message and Comment both send a message to be displayed in the instrument status window on the Mac. Which message to send is defined by the Miscellaneous (MISC) field. Messages contain instructions for the operator and comments contain informative descriptions. They are used in maintenance and test procedures such as bottle changes, sensor calibrations, and leak tests. Available messages and comments are listed below – some are reserved for possible future needs:

Messages List (for Send Message function 269):

“Place new 2g. amidites on synthesizer.” // Msg. 0

"Select 'resume' to continue.” // Msg. 1

“Auto-dilute procedure complete.” // Msg. 2

“Replace Amidites, Then Select Resume.” // Msg. 3

“Place empty columns in table pos. 1-9.” // Msg. 4

“ ” // Msg. 5

“Empty all bottles except Acetonitrile.” // Msg. 6




“Change reagent bottle now.” // Msg. 10

“Verify the needle is down in well 1.” // Msg. 11




“Verify the needle is at left waste.” // Msg. 15

“Verify the needle is at right waste.” // Msg. 16

“Verify that all regs display 5 psi.” // Msg. 17

“System Message 18.” // Msg. 18

“System Message 19.” // Msg. 19

Comments List (or Comment function 270):

“ACN (Reg 4)” // comment 0

“Tet & Bases (Reg 9)”

“NMI, AC2O & I2 (Reg 8)”

“TCA + Acetic Acid (Reg 2)”

“Syn Col A (Reg 7)”

“Syn Col B (Reg 7)” // comment 5

“Syn Col C (Reg 7)”


“Syn VB's (Reg 7)”

“Clv VB's & Bot Depro (Reg 10)”

“Clv Cols (Reg 10)”

“Depro Coils (Reg 1)” // comment 10

“Depro Coil B (Reg 1)”

“Depro Coil C (Reg 1)”

“NH4OH + TEAA (Reg 5)”

“Pur VB's & Up Depro (Reg3)”

“Pur Col A (Reg 3)” // comment 15

“Pur Col B (Reg 3)”

“Pur Col C (Reg 3)”

“TEAA (Reg 5)”

“TFA + 20% Acn (Reg 6)”

“DI (Reg 5)” // comment 20

“20% ACN (Reg 6)”

“UV (Reg 3)”

“Vent Amidite v76 (Reg 9)”

“Vent NH4OH v74 (Reg 5)”

“Test(s) completed.” // comment 25

“Message #26”

“Message #27”

“Message #28”

“Message #29”

271 Begin of Cycleand

272 End of Cycle

These functions serve only to mark the beginning and ending steps of a cycle or procedure.

273 Begin Loopand

274 End Loop

These functions mark the beginning and ending boundaries of a block of steps that will be executed repeatedly. The number of times that the steps will be repeated, the loop counter, is defined in the Miscellaneous (MISC) field of the Begin Loop step.

The block of steps within a loop will always be executed at least once, even if the Begin Loop parameter is zero. This is because it is the End Loop function at the end of the block that decides whether the steps have been repeated enough times to drop out of the loop or not. If not, the loop counter is decrements by 1 and the loop is repeated, starting with the first step after Begin Loop. When all loops are done, the cycle continues with the step after the End Loop step. Cycles must always have a matching number of Begin and End Loop steps.


Loops may be nested, that is, one or more loops may appear inside another loop. Loops may be nested up to ten levels deep. An example of loop nesting to two levels is outlined here:

Begin Loop 3 outer loop

[Steps to execute 3 times]

Begin Loop 5 inner loop

[Steps to execute 15 times] note: 15 = 3 x 5

End Loop end inner loop

End Loop end outer loop

When loops are nested, it is the loop countdown counter for the outermost loop that is displayed in the Chemistry Monitor window. For the above example, the counter displayed would be the one declared by the first Begin Loop step which would count down from the initial value of 3.

275 Start Here This function marks the initial cycle step for the first synthesis cycle only. Normally, a synthesis cycle begins with the coupling of a new base to the oligo, proceeds through capping and oxidation, and then detritylates in preparation for another base addition at the start of the next cycle. In the first synthesis cycle, the 3' base is already attached to the support so the appropriate point to begin this cycle is with the initial detritylation.

276 Exit DMT On This function executes only on the last synthesis cycle of any given column and it only affects sequences that are being synthesized “trityl on” (i.e., purified oligos or “trityl on” crudes). Its purpose is to prevent oligos from being detritylated on their final synthesis cycle when no further base additions are forthcoming.

The effect of this function is to deactivate the column(s) that are just concluding synthesis in time to avoid a final detritylation. Further, continuing synthesis on columns making longer oligos will have no effect on those columns that have already finished. Oligos being synthesized “trityl off” have their columns deactivated at Cycle End.


277 If Pur Col A The discussion below the table applies to all the functions listed in the table.

The functions listed above are all used to build a programming construct generally referred to as an if statement. The basic purpose of an if statement is to execute a series of steps only if a certain condition is true.

The minimal form of an if statement is:

If

steps to do if true

EndIf

continue on.

A more complex variation is:

If

steps to do if true

Else

steps to do if false

EndIf

continue on.

If statements may be nested to any number of levels (see function 273, Begin Loop) but only one End If is needed with many If's:

If

steps to do if true

If

steps to do if also true

EndIf

continue on.

If the test condition is not met (is false) then the cycle skips the steps between the If function and the Else or EndIf function, whichever occurs first, and continues executing from that point. If the test condition is true, then the steps immediately after the If function are executed and any steps between the Else step (if there is one) and the EndIf are skipped. The Else and EndIf functions act as markers in the cycle to tell the If functions what steps to skip.

The If functions in this particular group all involve the status of column activity. Most likely, these functions would be used only in purification cycles. The If Pur Col functions can be used to select certain cycle steps for execution only if the oligo for a given column is to be purified. Similarly, the If Crude Col functions can be used to select certain cycle steps for execution only if the column is handling a crude oligo.

Instead of operating based on the status of just one column, If Any Act Cols provides a means to conditionally execute steps for a group of columns involved with purifying

277 If Pur Col A to 279 If Pur Col C

280 Else

281 EndIf

291 If Any Act Cols

297 If Crude Col A to 299 If Crude Col C


oligos. After using function 282, Select Pur Cols, only those columns that remain active will be affected by the steps between If Any Act Cols and EndIf.

282 Select PurCols


The Select /Engage/Revive functions are all designed to change the status of which columns are active and which are not. Normally a column is active if it is processing an oligo, either crude or purified, and inactive if it is not. Column active status is initialized automatically at the beginning of each cycle for the set of oligos being processed during that cycle.

Select Pur Cols will deactivate any columns that are processing crude oligos and leave only those columns active that are processing purified oligos. Using this function in conjunction with If Any Act Cols provides a convenient means to restrict purification activities to just those columns that need them.

Engage All Cols ensures that all columns are active, even if they are not all currently processing oligos. One use for this feature is to guarantee that all of the coils are cleaned as needed. Otherwise, if a single oligo is being cleaved by the current cycle, only the coil for that oligo would get cleaned—even if the previous cycle had “dirtied” all three coils by filling them with oligos to be deprotected.

Revive Act Cols will restore to active status any column that was set up for synthesis but that has since been deactivated. This mechanism may be used to reactivate columns that were turned off because their syntheses had been concluded previously. When used in conjunction with If Cyc Greater, this function can enable the execution of the “end procedure” on all synthesizing columns during the last synthesis of the longest oligo in a set of three. This is useful for creating cycles to manage special additions at the 5´ end, i.e., extra washing after dye labelling or extra detritylation for certain modified aminos.

Revive Act Cols may also be used to reactivate columns that have been ended due to a jaw/block leak test failure or by the use of the End Row SCP/123 (see function number 229 above). By using this function in the purification cycle, oligos in the coils may be recovered even though the jaw/block leak test has failed.

Select Act Cols undoes the effects of the other three related functions by properly reselecting only normally active columns, i.e. just those columns actually processing oligos, regardless of type. If Select Pur Cols has turned off any columns processing crude oligos, this function will reactivate those columns. Similarly, columns will be deactivated if they are columns that have been reactivated by Revive Act Cols or columns without oligos that have been activated by Engage All Cols.

282 Select Pur Cols

283 Select Act Cols

300 Engage All Cols

319 Revive Act Cols


284 Clv Wants Depro


These three functions are used by cleavage and purification cycles to arbitrate between the two systems regarding the shared ownership of the deprotection coils.

Pur Owns Depro is executed early in the purification cycle to declare that the purification system has control of the coils. Once the deprotected oligos are transferred out of the coils into the purification vessels, the purification cycle executes Clv Owns Depro to surrender control of the coils to the cleavage system.

The cleavage cycle is responsible for cleaning out any residue from previously deprotected oligos before transferring in the most recently cleaved oligos. A Clv Wants Depro step is placed in the cleavage cycle immediately before this cleaning begins. The cleavage cycle waits at this step as long as the purification system retains ownership of the coils. Control of the coils is eventually transferred to cleavage (Clv Owns Depro) after the purification cycle has removed the previous set of deprotected oligos from the coils. At this point, the cleavage cycle moves on to clean the coils, transfer in the current set of oligos, and start deprotection.

Since there is no purification cycle running during the first cleavage cycle, Clv Owns Depro is executed in the begin procedure to ensure that the cleavage system is in possession of the coils when the first set of oligos in a run is ready for deprotection.

287 Go Sub The discussion below the table applies to all the functions listed in the table.

The set of functions above is involved with subroutines. A subroutine is a set of steps that accomplishes a particular task, such as filling or draining a set of vessels, performing a detritylation, or cleaning a transfer path. A cycle or procedure may have many subroutines.

Subroutines are always placed at the end of the cycle, after a Goto End step. The Goto End step is a marker that says to ignore all of the steps in the subroutines that follow and jump directly to the Cycle End step. Effectively, the Goto End step is the last step of the main cycle.

The Sub Label function marks the beginning of a subroutine. The Miscellaneous (MISC) field in the Sub Label step uniquely identifies a specific subroutine. All of the subroutines within a given cycle or procedure must have different numbers. Subroutines are only recognized within their cycle so the same Sub Label may be applied to different subroutines within different cycles.

By using Go Sub, a subroutine may be “called” from anywhere in the main cycle to do its job. Calling a subroutine means that cycle execution jumps from the main cycle down to the first step of the subroutine and begins executing the steps following the

284 Clv Wants Depro

285 Pur Owns Depro

286 Clv Owns Depro

287 Go Sub

288 Return Sub

289 Sub Label

290 Goto End


Sub Label. The Miscellaneous field in a Go Sub step identifies the corresponding Sub Label step to jump to. Subroutines may not be called from other subroutines.

Return Sub marks the end of a subroutine. The steps in between the Sub Label and Return Sub steps constitute the working body of the subroutine. When a Return Sub step is reached, execution jumps back up to the main cycle and resumes at the step following the Go Sub step that called the subroutine. Column activity status may be adjusted by Return Sub (see Go Sub A/B/C below).

Subroutines are useful when the work of the subroutine must be done a number of times in different places in a cycle. With subroutines, only one copy of the steps required to do a given job needs to be created. Cycles may be written with fewer steps because every time the same steps recur in a cycle they can be replaced by a single Go Sub call. Also, if a subroutine's process ever needs alteration, changes need only be made in just one place instead of in several locations.

291 If Any ActCols

See preceding discussion of function 277, If Pur Col A, et al.

292 Go Sub A to

294 Go Sub C

These functions, called conditional Go Sub's, are a variation of the standard Go Sub function with a Select function twist (see function 282, Select Pur Cols). Each of these functions executes a subroutine call to the Sub Label step with a matching Miscellaneous (MISC) field value. However, this set of Go Sub's will execute the subroutine call only if their corresponding column is active.

A second feature of conditional Go Sub's is that when the subroutine is called, any other active columns are deactivated for the duration of the subroutine's execution. When a conditional Go Sub call is used, the Return Sub function automatically restores column activity status to what it was when the subroutine call was made. This status may be all columns active, only purification columns active, etc. (see function 282, Select Pur Cols, et al.)

Conditional Go Sub's take advantage of the fact that almost all column delivery functions come in sets consisting of one parallel version of the function and three serial, column-specific versions (e.g., ACN to Syn Cols and ACN to Syn Col A, B, or C). When only one column is active, the parallel version of a function will behave exactly like the serial version for that active column. If a subroutine is written using parallel functions and then called conditionally, it will act like a subroutine written for one specific column.

Conditional subroutine calls may be used to help minimize the total number of cycle steps, simplify cycle updates, and maximize the uniformity of cycle execution across columns.


295 If First Cycle The discussion below the table applies to all the functions listed in the table.

This is another category of If functions which must be paired with an Endif as discussed above (see function 277, If Pur Col A). These functions are only valid in a synthesis cycle. They cause a block of steps to be conditionally executed or skipped over based on the number of the cycle currently executing.

The block of steps between If First Cycle and its corresponding Else or Endif step will only be executed if the current cycle in progress is the first cycle of synthesis. Similarly, the block of steps between If Last Cycle and its corresponding Else or Endif step will only be executed if the current cycle in progress is the last cycle of the longest oligo being synthesized in a given row. These functions are useful for creating “begin -” and “end procedures” for synthesis cycles which may be used for instrument housekeeping purposes or to implement special cycle variations. When implementing special 5´ monomer cycles, If Last Cycle will usually be used in conjunction with Revive Act Cols (see the preceding discussion of function 282, Select Pur Cols, et al).

The If Cyc Greater function enables the synthesis cycle to modify or adapt its activities as the oligo it creates gets longer. The Miscellaneous (MISC) field is used to define the cycle number at which synthesis will begin to execute additional steps. By nesting If Cyc Greater steps (see function 277, If Pur Col A), new activities may be gradually incorporated into the cycle as the oligo reaches new milestone lengths.

Having an adaptive cycle removes the requirement to have multiple cycles for different length oligos. It also allows the chemistry to perform optimally at each oligo length instead of attempting to strike a balance between the activity required during early cycles and that required later on. This capability facilitates minimizing cycle time and maximizing cycle performance.

297 If Crude Col Ato

299 If Crude Col C

See preceding discussion of function 277, If Pur Col A, et al.

300 Engage AllCols

See the preceding discussion of function 282, Select Pur Cols, et al.

316 If Cyc Greater See the preceding discussion of function 295, If First Cycle, et al.

317 Save Reg Presand

318 RestoreRegPres

Both of these functions use the Miscellaneous (MISC) field to declare the number of the regulator involved. Save Reg Pres will save the regulator pressure setting value of the specified regulator. Restore RegPres resets the pressure setpoint of the regulator to the value saved by Save Reg Pres. Regulator pressures may be saved and restored by subroutines that alter the regulator's pressure during their execution.

319 Revive ActCols

See the preceding discussion of function 282, Select Pur Cols, et al.

295 If First Cycle

296 If Last Cycle

316 If Cyc Greater


320 Check SnsrCal

This function checks that the wet sensor calibration values are at least twice the dry values for all sensors and issues a Critical Message if any sensor calibration fails to meet this standard. Check Snsr Cal is used in the standard begin procedure and in the sensor calibration procedure(s).

325 Jaw Test Times See the preceding discussion of function 232, Syn Jaw Close, et al.

329 Begin Leak Testand

330 End Leak Test

The Leak Test functions mark the begin and end of a pressure/leak test. A Wait step is placed between these two functions to define the pressure drop time for the test.

The Time field in a Begin Leak Test step defines how much time to use taking an initial baseline pressure average. The Miscellaneous (MISC) field defines which regulator pressure is being tested.

The Time field in an End Leak Test step defines how much time to use taking a final average pressure reading. The Miscellaneous field defines the pass/fail pressure drop threshold for the test in 1/100's of a psi.

When the pass/fail threshold is less than 1 psi, a leak test is being performed and the test passes when the pressure drop from initial to final reading is less than the threshold. When the pass/fail threshold is greater than 1 psi, a vent test is being performed and the test passes when the pressure drop is greater than the threshold.

396 Time Stamp This function enters the ABI 3948 System’s current time-of-day into the Microphone log. This time stamp is tagged with the number in the Miscellaneous (MISC) field. The convention is that time stamps for cleavage are tagged with the numbers 20 through 29 and the time stamps for purification are tagged 30 through 39.

Time stamps were created as an aid to cycle development so that timing information for the various parts of any cycle might be easily tracked. When a time stamp is logged, the time of the previous time stamp for the given chemistry controller is also reported. This makes it easier to calculate the duration between the time stamps bracketing the start and end of some portion of a cycle.

401 SynUpr Wet 401to

500 UV Dry 500

The last 100 functions on the ABI 3948 System function list are user functions that can perform sensor-controlled deliveries. Like standard user functions, the valve list for these functions can be defined by the operator. These functions offer the additional advantage of having sensors pre-assigned to them. These sensors are engaged in the usual fashion by setting the SNS field in the step to YES. The particular sensor or set of sensors tied to a sensor user function is specified in the default function name. As with standard user functions, the function name may be edited as well as the valve list.

There are three components to the default names provided for sensor user functions. The first part of the default name identifies the sensor or sensors associated with the function. The second component spells out whether the function is “wet” or “dry”, that is, whether the function's sensor(s) trip upon detecting wetness (liquid delivery) or dryness (gas delivery, flush or back flush). In keeping with the convention for standard user functions, the function number is included as the final component of the default function name.



Note Parallel sensor functions do not have valve lists of their own but use the valve lists of the associated serial sensor functions. Valve assignments must be made properly for all serial functions of a given type for the parallel sensor function to work or work properly. The

SynUpr, SynMan, and SynLow refer respectively to the upper synthesis sensors numbered 1 to 3, the synthesis manifold sensors numbered 22- to 24, and the lower synthesis sensors numbered 4 to 6. Sensors 7 to 9 are the Clv sensors, sensors 10 to 12 are the Coil sensors, and sensors 14 to 16 are the Pur sensors. Sensor 21 is the UV sensor. (See the Plumbing Diagram in Appendix E to verify which sensors are associated with which columns. In some instances, the column alphabetic order and sensor numeric order are reversed.)


This grouping of sensor functions into sets is crucial to the operation of parallel functions. Parallel functions do not use their own valve lists to execute, they use the valve lists of their associated serial functions instead. In this way, parallel functions will turn on only the valves for active columns by merging together the valve list for each active column's associated serial function and excluding the valve lists of functions associated with inactive columns. When one column finishes its delivery before the others, its single set of valves is deactivated. Any other active columns' valves will remain on until their sensors trigger.

User definable functions operate according to the same scheme. In order to program the valve list for a parallel user function, the appropriate valve lists for each associated serial function must be entered. The value to also entering the complete, three-column valve set into the parallel function's valve list is one of documentation: the entire valve list may then be viewed under one function in Manual Control.

Cycles, Procedures, and System Messages C-1

Cycles, Procedures, and System Messages C

In This Appendix

Introduction This appendix contains listings of the standard synthesis, cleavage/deprotection, and purification cycles for use in understanding ABI 3948 System chemistry.

Topics Covered This appendix contains the following topics:

Topic See page

ABI 3948 System Chemistry C-2

Overview C-2

The Standard Synthesis Cycle C-2

Synthesis Cycle Summary C-2

Synthesis Cycle Listing C-4

The Standard Cleavage/Deprotection Cycle C-8

Cleavage/Deprotection Cycle Summary C-8

Cleavage/Deprotection Cycle Listing C-11

The Standard Purification Cycle C-17

Purification Cycle Summary C-17

Purification Cycle Listing C-22

Procedures C-31

General C-31

Edit Begin Procedure View C-31

Edit End Procedure View C-33

Edit Bottle Procedure View C-35

Miscellaneous Procedures C-36

System Messages C-37

Instrument Status and Message Indicators C-37

Types of Messages/Categories of Messages/Conventions C-37

Regular Reports Messages C-38

Critical Messages C-40

Microphone-only Messages C-40

Hierarchical Messages C-41

C

C-2 Cycles, Procedures, and System Messages

3948 Chemistry

Overview Chemistry Protocols

Chemistry in the ABI 3948 System is performed by assigning a protocol to each oligonucleotide to be produced. A protocol consists of three cycles to perform the three types of chemistry: synthesis, cleavage/deprotection, and purification. Each cycle is a series of functions which operate the valves and perform the other actions needed to produce an oligonucleotide.

Functions in Cycles

Functions are arranged in a particular order to create a cycle, which performs an operation. One Synthesis cycle, for example, contains all the functions necessary for one base addition. The functions in each standard cycle have optimized delivery times to ensure completion of each chemical reaction with minimal reagent consumption and minimal unwanted side reactions.

User Cycles

Besides the standard cycles, you can develop up to 34 additional cycles, 18 for synthesis, 8 for Cleavage/Deprotection, and 8 for Purification. Although the standard cycles cannot be changed or deleted, they can be copied into one of the user cycle locations and edited with the Cycle Editor to provide the basis for a new user cycle.

Synthesis Scale

The ABI 3948 System synthesizes at one scale only. Synthesis of a 25-mer typically yields 8.0-12 ODU of crude oligonucleotide, which when purified may equal 0.5 to 4 ODU.


The Standard Synthesis Cycle

Synthesis CycleSummary

The Synthesis cycle processes/reactions needed for synthesis are illustrated in the following flow chart (Figure C-1) and the step numbers corresponding to these activities are listed in Table C-1. Table C-2 contains a listing of the standard synthesis cycle.

Figure C-1 Synthesis Cycle Flow Chart


Table C-1 Summary of Synthesis Cycle

Step Range Description

47 - 52 Start Here, Leak Test

� Close synthesis jaw, begin leak test and check for pass or fail.

� If leak test passes, go to if first cycle.

� If leak test fails, go to cycle end.

53 - 67 ACN Wash

� If first cycle, give columns extra ACN wash.

� Normal column(s) ACN wash.

� Normal column(s) ACN wash

� If last cycle, synthesis module cleanup (Go Sub 3).

68 - 86 Detritylation

� Exit DMT On, for “purified or trityl on crude’s” only.

� This function executes only on the last synthesis cycle for each column.

� 7 loop of detritylation (Go Sub 3).

� If Cyc Greater, additional detritylation for longer oligos (Go Sub 2).

� If first cycle, additional detritylation for support bound nucleoside (Go Sub 2).

87 - 100- 44 Trityl Wash

� Wash and flush to trityl waste with ACN.

� If last cycle, execute synthesis module cleanup (Go Sub 3) in the case of “trityl off crudes”.

1 - 19 Begin of Cycle, Base+Tet (Coupling)

� Tetrazole and Base+Tet delivery.

� Coupling push/wait loop

� If Cyc Greater, extend coupling for longer oligos.

20 - 31 Capping

� Deliver capping reagents to manifold sensors.

� Capping push/wait loop

32 - 46 Oxidation

� Deliver Iodine to manifold sensors.

� Iodine push/wait loop


Synthesis CycleListing

The annotated listing below is provided to aid you in understanding ABI 3948 System synthesis:

Table C-2 Standard Synthesis Cycle (v4.20)

STEP FUNCTION NUM TIME MISC SNS SAFE Comments

1) Begin of Cycle 271 0.0 0 Yes Yes Marks beginning of cycle

2) SynUprBlk Flush 105 2.0 0 No Yes Flush out upper synthesis block

3) TET to Syn Cols 48 5.0 0 Yes Yes Del. Ieading TET slug to lower sensors

4) B+ TET to Syns 1 0.0 0 Yes Yes Push TET to manifold sensors w/Base+Tet

5) ACN to SynWaste 47 2.0 0 No Yes Rinse and prime lower block w/ACN

6) ACN to Syn Cols 43 15.0 0 Yes Yes Push Base+TET to upper sensor w/ACN

7) Begin Loop 273 0.0 5 No Yes Begin coupling push/wait loop

8) Coupling Wait 26 8.0 0 No Yes Wait for Coupling (from B+TET table)

9) If Cyc Greater 316 0.0 60 No Yes iif after cycle/base 6O,

10) Wait 259 2.0 0 No Yes extend wait for coupling

11) If Cyc Greater 316 0.0 75 No Yes iif after cycle/base 75,

12) Wait 259 2.0 0 No Yes further extend wait for coupling

13) If Cyc Greater 316 0.0 90 No Yes iif after cycle/base 9O,

14) Wait 259 2.0 0 No Yes further extend wait for coupling

15) Endif 281 0.0 0 No Yes End of all "if cyc greater"

16) ACN to SynCol A 44 0.3 999 No Yes Push amidite to column A w/ACN or wait

17) ACN to SynCol B 45 0.3 999 No Yes Push amidite to column B w/ACN or wait

18) ACN to SynCol C 46 0.3 999 No Yes Push amidite to column C w/ACN or wait

19) End Loop 274 0.0 0 No Yes End coupling loop

20) SynLowBlk Flush 65 1.0 0 No Yes Flush out lower synthesis block

21) Go Sub 287 0.0 1 No Yes Flush columns/lines/blocks

22) CAP to Syn Cols 27 15.0 0 Yes Yes Deliver CAP to manifold sensors


24) ACN to Syn Cols 43 15.0 0 Yes Yes Push CAP to upper sensors w/ACN

25) Wait 259 1.0 0 No Yes Initial capping wait, all columns

26) Begin Loop 273 0.0 5 No Yes Begin capping push/wait loop

27) ACN to SynCol A 44 0.3 999 No Yes Push CAP to column A w/ACN or wait

28) ACN to SynCol B 45 0.3 999 No Yes Push CAP to column B w/ACN or wait

29) ACN to SynCol C 46 0.3 999 No Yes Push CAP to column C w/ACN or wait

30) Wait 259 1.4 0 No Yes Wait for capping, all columns

31) End Loop 274 0.0 0 No Yes End capping push/wait loop

32) ACN to SynWaste 47 1.0 0 No Yes Rinse lower block w/ACN



35) IODINE to Syns 33 15.0 0 Yes Yes Deliver iodine to manifold sensors


37) ACN to Syn Cols 43 15.0 0 Yes Yes Push iodine to upper sensors w/ACN

38) Wait 259 1.0 0 No Yes Initial oxidation wait, all columns


39) Begin Loop 273 0.0 5 No Yes Begin oxidation push/wait loop

40) ACN to SynCol A 44 0.3 999 No Yes Push iodine to column A w/ACN or wait

41) ACN to SynCol B 45 0.3 999 No Yes push iodine to column B w/ACN or wait

42) ACN to SynCol C 46 0.3 999 No Yes push iodine to column C w/ACN or wait

43) Wait 259 1.4 0 No Yes Wait for oxidation, all columns

44) End Loop 274 0.0 0 No Yes End oxidation push/wait loop

45) SynLowBlk Flush 65 1.5 0 No Yes Fiush out lower synthesis block

46) Go Sub 287 0.0 1 No Yes Flush columns/lines/blocks .

47) Start Here 275 0.0 0 No Yes First cycle begins here, detritylation

48) Syn Jaw Close 232 0.0 0 No Yes Close syn jaw, start syn module leak test

49) If Any Act Cols 291 0.0 0 No Yes Test for syn module leak test failure

50) Else 280 0.0 0 No Yes No action if passes

51) Goto End 290 0.0 0 No Yes Skip to end of cycle on failed leak test

52) Endif 281 0.0 0 No Yes End of syn module leak test

53) If First Cycle 295 0.0 0 No Yes First cycle only, lines may be wet



56) Flush Syn Col A 54 3.0 0 No Yes Flush column A

57) Flush Syn Col B 55 3.0 0 No Yes Flush column B

58) Flush Syn Col C 56 3.0 0 No Yes Flush column C


60) Go Sub 287 0.0 4 No Yes Deliver ACN to upper sensors


62) Endif 281 0.0 0 No Yes End first cycle line clean

63) Go Sub 287 0.0 4 No Yes Deliver ACN to upper sensors


65) If Last Cycle 296 0.0 0 No Yes If last cycle, clean lines/blocks

66) Go Sub 287 0.0 3 No Yes Last cycle wash cola/lines/blocks

67) Endif 281 0.0 0 No Yes End if last cycle line/block clean

68) Exit DMT On 276 0.0 0 No Yes End here if trityl on


70) TCA to Syn Cols 38 4.0 0 No No Start split delivery of TCAto columns

71) TCA to Syn Cols 38 15.0 0 Yes No Finish split delivery of TCA to sensors

72) Begin Loop 273 0.0 7 No No Begin detritylation loop

73) Go Sub 287 0.0 2 No No Serial TCA (detritylation) push

74) End Loop 274 0.0 0 No No End detritylation loop

75) If Cyc Greater 316 0.0 60 No No if after cycle/base 60, additional TCA push





Table C-2 Standard Synthesis Cycle (v4.20) (continued)




81) Endif 281 0.0 0 No No End of all "if cyc greater tests

82) If First Cycle 295 0.0 0 No No First cycle only, extra detritylation

83) Begin Loop 273 0.0 3 No No Begin first cycle oniv detritylation loop


85) End Loop 274 0.0 0 No No End first cycle only detritylation loop

86) Endif 281 0.0 0 No No End "if first cycle"

87) LowTrityl Flush 106 1.0 0 No No Flush lower block to halogenated wate

88) ACN to Trityl 108 2.0 0 No No Rinse lower block to halogenated wate

89) LowTrityl Flush 106 2.0 0 No No Flush lower block to halogenated wate

90) Waste to Trityl 109 0.0 999 No No Redirect v22 waste to v23 halo waste columns/lines/blocks

91) Go Sub 287 0.0 1 No No Flush columns/lines/blocks .

92) Go Sub 287 0.0 4 No No Deliver ACN to upper sensors

93) Go Sub 287 0.0 1 No No Flush columns/lines/blocks

94) Flush Syn Col A 54 2.0 0 No Yes Flush column A, extra dry step

95) Flush Syn Col B 55 2.0 0 No Yes Flush column B, extra dry step

96) Flush Syn Col C 56 2.0 0 No Yes Flush column C, extra dry step

97) Waste to Trityl 109 0.0 0 No Yes Restore v22 waste output

98) If Last Cycle 296 0.0 0 No Yes If last cycle, wash of cola/lines/blocks

99) Go Sub 287 0.0 3 No Yes Last cycle wash cola/lines/blocks

100) Endif 281 0.0 0 No Yes End if last cycle

101) Goto End 290 0.0 0 No Yes Skip subroutines to end of synthesis cycle

102) Sub Label 289 0.0 1 No No Flush columns/lines/blocks

103) SynLowBlk Flush 65 2.0 0 No No Flush out lower synthesis block

104) Flush Syn Cols 53 15.0 0 Yes No Flush lines thru columns to upper sensors

105) Flush Syn Cols 53 1.0 0 No No Short flush above upper sensors

106) Return Sub 288 0.0 0 No No End of Sub 1

107) Sub Label 289 0.0 2 No No Serial TCA (detritylation push

108) TCA to SynCol C 41 1.0 999 No No Push TCA to column C

109) TCA to SynCol B 40 1.0 999 No No Push TCA to column B

110) TCA to SynCol A 39 1.0 999 No No Push TCA to column A


112) Sub Label 289 0.0 3 No Yes Last cycle wash cola/lines/blocks

113) Revive Act Cols 319 0.0 0 No Yes Make all starting columns active again

114) LowTrityl Flush 106 10.0 0 No Yes Flush lower block to halogenated waste


116) ACN to Syn Cols 43 2.0 0 No Yes Start split delivery of ACN

117) ACN to Syn Cols 43 15.0 0 Yes Yes Finish split delivery of ACN to sensors

118) Flush Syn Col A 54 20.0 0 No Yes Flush colum A to dryness

119) Flush Syn Col B 55 20.0 0 No Yes Flush colum B to dryness




120) Flush Syn Col C 56 20.0 0 No Yes Flush colum C to dryness



123) Select Act Cols 283 0.0 0 No Yes Switch off all previously completed columns

124) Return Sub 288 0.0 0 No Yes End of Sub3

125) Sub Label 289 0.0 4 No No Deliver ACN to upper sensors

126) ACN to Syn Cols 43 4.0 0 No No Start split delivery of ACN

127) ACN to Syn Cols 43 15.0 0 Yes No Finish split delivery of ACN to sensors

128) ACN to Syn Cols 43 0.5 0 No No Short ACN push past sensors


130) End of Cycle 272 0.0 0 Yes Yes Marks end of cycle and all sub routines




The Standard Cleavage/Deprotection Cycle

Cleavage/Deprotection Cycle

Summary

The Cleavage/Deprotection cycle processes/reactions needed for oligo production are illustrated in the following flow chart (Figure C-2) and the step numbers corresponding to these activities are listed in Table C-3. Table C-4 contains a listing of the standard cleavage/deprotection cycle:

Figure C-2 Cleavage/Deprotection Cycle Flow Chart


Table C-3 Summary of Cleavage/Deprotection Cycle


1 - 10 Test for Active Columns

� Test oligos that have failed synthesis module leak test (failed columns will not be active.

� If synthesis module failed leak test, then (a) wait for synthesis and purification modules to complete leak testing, (b) wait until the deprotection coils are free and (c) clean up the cleavage module plumbing (Go Sub 9).(Go to cycle end).

� If columns Active, then close the synthesis jaw.

11 - 17 Leak Test, Start Here if Columns are Active

� Close the cleavage jaw, begin leak test and check for pass or fail.

� If cleavage module failed, the (a) wait until the deprotection coils are free and (b) clean up the cleavage module plumbing (Go Sub 9). (Go to cycle end).

� If cleavage module passed leak test, then start normal cleavage cycle.

18 - 33 Cleaning of Cleavage Vessels, Prep for Crude Oligos

� Water wash and flush of lower cleavage block.

� Water wash, flush and drying of cleavage columns, vessels and lines.

34 - 59 Ammonia Cleave of Oligo from the Support

� Start Cleavage by delivering ammonia to the upper cleavage sensors. Rinse ammonia from the lower cleavage block. Wait while cleaving.

� Set Regulator 10 for the first push of fresh ammonia over the columns (Go Sub 1). Wait while cleaving.

� Begin cleavage loop, push fresh ammonia over the columns (Go Sub 1) and wait while cleaving.

60 Wait for Purification to Surrender the Deprotection Coils to Cleavage

� Cleavage wants the deprotection coils


61 - 74 Prepare and Transfer Oligos from the Cleavage Vessels to the Deprotection Coils

� Set coil temperature for the transfer INTO the coils. Wash the transfer line, coils and cleavage blocks.

� Set the ramping function parameters.

� Gently consolidate sample into cleavage vessels.

� Ramping transfer into coils from cleavage vessels.

75 Start Deprotection Heater

� Start the clock for deprotection time (set in preferences) once the target temperatures reached.

76 - 93 Column Strip of Uncleaved Product

� Start column strip, deliver ammonia to upper cleavage sensors. Rinse ammonia from the lower cleavage block. Wait while stripping.

� Set regulator 10 for the first push of fresh ammonia over the columns (Go Sub 1). Wait while stripping.

� Begin column stripping loop, push fresh ammonia over the columns (Go Sub 1) and wait while stripping.

94 - 107 Wash, Drain and Dry Cleavage Module

� Drain an dry cleavage module.

� Water and ACN washes of columns, vessels and lines.

� Drain an dry cleavage module.

Table C-3 Summary of Cleavage/Deprotection Cycle (continued)



Cleavage/Deprotection Cycle

Listing

The annotated listing below is provided to aid you in understanding ABI 3948 System cleavage and deprotection:

Table C-4 Standard Cleavage Cycle (v4.20)


1) Begin of Cycle 271 0.0 0 No Yes Marks beginning of cycle

2) If Any Act Cols 291 0.0 0 No Yes Test for oligos that failed in synthesis

3) Else 280 0.0 0 No No If ok do nothing, else clean civ plumbing

4) Wait 259 60.0 0 No Yes Wait for syn/pur module leak testing

5) Wait 259 60.0 0 No No Wait for syn/pur module leak testing

6) Wait 259 60.0 0 No Yes Wait for syn/pur module leak testing

7) Clv Wants Depro 284 0.0 0 No Yes Wait until deprotection coils are free

8) Go Sub 287 0.0 9 No Yes Drain/wash/dry of coils & depro blocks

9) Goto End 290 0.0 0 No Yes Skip over subroutines to cycle end

10) Endif 281 0.0 0 No Yes To reach this step, columns are active

11) Clv Jaw Close 234 0.0 0 No Yes Close civ jaw, start civ module leak test

12) If Any Act Cols 291 0.0 0 No Yes Test for module leak test failure

13) Else 280 0.0 0 No Yes No action if test passes

14) Clv Wants Depro 284 0.0 0 No Yes Leak test failed, wait for depro coils

15) Go Sub 287 0.0 9 No Yes Drain/wash/dry of coils & blocks


17) Endif 281 0.0 0 No Yes To reach this step, the leak test passed

18) Time Stamp 396 0.0 20 No Yes Record current time

19) Set Pres Reg 10 310 0.0 12 No Yes Set regulator 10 to 12 psi

20) Clv Blk Flush 144 5.0 0 No Yes Flush lower cleavage blocks, may be wet

21) H2O to ClvWaste 130 5.0 0 No Yes Wash lower cleavage blocks

22) Clv Blk Flush 144 5.0 0 No Yes Flush lower cleavane blocks

23) Begin Loop 273 0.0 2 No Yes Begin vessel/column/line water wash

24) Gas to Clv Cols 121 8.0 0 No Yes Dry vessels/columns/lines

25) Gas to ClvCol A 122 3.0 0 No Yes Dry vessel/column/line A

26) Gas to ClvCol B 123 3.0 0 No Yes Drv vessel/column/line B

27) Gas to ClvCol C 124 3.0 0 No Yes Dry vessel/column/line C

28) Go Sub 287 0.0 7 No Yes Delivery of water to sensor

29) Go Sub 287 0.0 6 No Yes Serial 5 psi gas bubble of vessel


31) Go Sub 287 0.0 4 No Yes Drain vessel/column/line

32) Go Sub 287 0.0 5 No Yes Dry vessels/columns/lines

33) End Loop 274 0.0 0 No Yes End vessel/column line wash


35) Go Sub 287 0.0 3 No Yes Deliver ammonia to start cleavage

36) Clv Blk Flush 144 10.0 0 No Yes Flush ammonia from lower civ blocks

37) Dep Xfer Rinse 151 5.0 0 No Yes Wash ammonia from lower civ blocks


38) Clv Blk Flush 144 10.0 0 No Yes Dry lower cleavage blocks

39) Wait 259 60.0 0 No Yes Wait while cleaving




43) Go Sub 287 0.0 1 No Yes Push fresh NH4 over cols for cleavage


45) Wait 259 60.0 0 No Yes Wait while cleavinq



48) Begin Loop 273 0.0 5 No Yes Begin cleavage loop

49) Go Sub 287 0.0 1 No Yes Push fresh NH4 over cols for cleavage








57) Wait 259 60.0 0 No Yes Wait while cleavina

58) End Loop 274 0.0 0 No Yes End cleavage loop


60) Clv Wants Depro 284 0.0 0 No Yes Wait until deprotection coils are free


62) Set Coil Temp 260 0.0 0 No Yes Temp for transfer into coils (set in prefs)

63) Go Sub 287 0.0 9 No Yes Drain/wash/dry of coils & blocks


65) Set Rmp Fxn Sns 265 0.0 10 No Yes Set ramp parms: # of total readings

66) Set Rmp Fxn Trp 266 0.0 9 No Yes Set ramp parms: # of readings to trip

67) Set Rmp Fxn Chk 267 0.0 7 No Yes Set ramp parms: # of readings to verify

68) Set Rmp Fxn Dly 268 0.0 50 No Yes Set ramp parms: # of ms to delay verify

69) Go Sub C 294 0.0 2 No Yes Prepare transfer to coil C

70) Xfer Clv Col C 143 12.0 10 Yes Yes Ramping transfer from vessel C to coil C

71) Go Sub B 293 0.0 2 No Yes Prepare transfer to coil B

72) Xfer Clv Col B 142 12.0 10 Yes Yes Ramping transfer from vessel B to coil B

73) Go Sub A 292 0.0 2 No Yes Prepare transfer to coil A

74) Xfer Clv Col A 141 12.0 10 Yes Yes Ramping transfer from vessel A to coil A

75) Start Depro Htr 170 0.0 0 No Yes Start depro heater (set in preferences)



78) Go Sub 287 0.0 3 No Yes Deliver ammonia for column strip

Table C-4 Standard Cleavage Cycle (v4.20) (continued)



79) Wait 259 30.0 0 No Yes Wait while stripping column



82) Ammonia toWaste 115 1.0 0 No Yes Prime block with ammonia

83) Ammonia to ClvA 112 1.0 0 No Yes Deliver ammonia to column A

84) Ammonia to ClvB 113 1.0 0 No Yes Deliver ammonia to column B

85) Ammonia to ClvC 114 1.0 0 No Yes Deliver ammonia to column C

86) Clv Blk Flush 144 2.0 0 No Yes Flush ammonia from lower civ blocks



89) Begin Loop 273 0.0 6 No Yes Begin column strip loop

90) Go Sub 287 0.0 1 No Yes Gas push (for strip)

91) Wait 259 30.0 0 No Yes Wait while stripping

92) Wait 259 60.0 0 No Yes Wait while stripping

93) End Loop 274 0.0 0 No Yes End column strip loop

94) Go Sub 287 0.0 4 No Yes Drain & dry vessel/column/line


96) Go Sub 287 0.0 7 No Yes Delivery of water to sensor

97) Go Sub 287 0.0 6 No Yes Serial 5 psi gas bubble of vessel

98) Go Sub 287 0.0 4 No Yes Drain vessel/column/line


100) Go Sub 287 0.0 8 No Yes 2:1 water/ACN vessel wash

101) Go Sub 287 0.0 4 No Yes Drain & dry vessel/column/line



104) Go Sub 287 0.0 4 No Yes Drain vessels/column/line




108) Sub Label 289 0.0 1 No Yes Gas push (for cleavage & strip)

109) Clv Blk Flush 144 1.0 0 No Yes Flush lower cleavage blocks

110) Press Line 138 2.0 0 No Yes Pressurize line to avoid-reverse flow

111) Gas to ClvCol A 122 0.6 0 No Yes Push of fresh NH4 over col A for strip


113) Press Line 138 2.0 0 No Yes Pressurize line to avoid reverse flow

114) Gas to ClvCol B 123 0.6 0 No Yes Push of fresh NH4 over col B for strip


116) Press Line 138 2.0 0 No Yes Pressurize line to avoid reverse flow

117) Gas to ClvCol C 124 0.6 0 No Yes Push of fresh NH4 over col C for strip

118) Return Sub 288 0.0 0 No Yes End of subroutine 1

119) Sub Label 289 0.0 2 No Yes Prepare transfer to coils AIB/C





121) Dep Xfer Rinse 151 3.0 0 No Yes Water rinse for transfer line

122) Dep Xfer Flush 152 20.0 0 No Yes Flush transfer line


124) DepUprBlk Rinse 154 3.0 0 No Yes Water rinse for upper depro block

125) DepUprBlk Flush 221 5.0 0 No Yes Flush upper depro block

126) DepLowBlk Flush 153 2.0 0 No Yes Flush lower depro block


128) Dep Xfer Flush 152 4.0 0 No Yes Reduce regulator 10 pressure

129) Gas to Clv Cols 121 12.0 0 No Yes Consolidate sample into vessels

130) Set Pres Reg 10 310 0.0 4 No Yes Starting transfer pressure

131) Dep Xfer Flush 152 2.0 0 No Yes Reduce regulator 10 pressure


133) Sub Label 289 0.0 3 No Yes Deliver ammonia for cleave/strip

134) Ammonia toWaste 115 1.0 0 No Yes Prime block w/ ammonia

135) Ammonia to Clvs 111 3.0 0 No Yes Start split delivery of NH4

136) Ammonia to Clvs 111 15.0 0 Yes Yes Finish split delivery of NH4 to sensors


138) Sub Label 289 0.0 4 No Yes Drain vessel/column/line


140) Back Flush Clvs 131 6.0 0 No Yes Wet sensors for all active vessels

141) Back Flush Clvs 131 45.0 0 Yes Yes Drain all vessels until sensors read dry

142) Back Flsh Clv A 132 6.0 0 No Yes Complete drain of vessel A

143) Back Flsh Clv B 133 6.0 0 No Yes Complete drain of vessel B

144) Back Flsh Clv C 134 6.0 0 No Yes Complete drain of vessel C

145) Begin Loop 273 0.0 2 No Yes Begin vessel drain/dry loop

146) Gas to Clv Cols 121 1.0 0 No Yes Reverse drying

147) Back Flush Clvs 131 4.0 0 No Yes Backflush drying

148) End Loop 274 0.0 0 No Yes End vessel drain/dry loop


150) Sub Label 289 0.0 5 No Yes Dry vessels/columns/lines


152) Begin Loop 273 0.0 2 No Yes Begin drain/dry loop

153) Back Flsh Clv A 132 5.5 0 No Yes Drain column A

154) Back Flsh Clv B 133 5.5 0 No Yes Drain column B

155) Back Flsh Clv C 134 5.5 0 No Yes Drain column C

156) End Loop 274 0.0 0 No Yes End drain/dry loop

157) Gas to Clv Cols 121 2.0 0 No Yes Reverse drying

158) Back Flush Clvs 131 8.0 0 No Yes Backflush drying


160) Sub Label 289 0.0 6 No Yes Serial 5 psi gas bubble of vessel





162) Gas to ClvCol A 122 8.0 0 No Yes Bubble vessel A

163) Gas to ClvCol B 123 8.0 0 No Yes Bubble vessel B

164) Gas to ClvCol C 124 8.0 0 No Yes Bubble vessel C


166) Sub Label 289 0.0 7 No Yes Delivery of water to sensor

167) H2O to ClvWaste 130 1.0 0 No Yes Prime lower cleavage blocks w/ water

168) H2O to Clv Cols 126 2.0 0 No Yes Start split delivery of water

169) H2O to Clv Cols 126 15.0 0 Yes Yes Finish split delivery of water to sensor


171) Sub Label 289 0.0 8 No Yes 2:1 water/ACN vessel wash


173) ACN to Clv Cols 116 20.0 0 Yes Yes Deliver ACN to sensors

174) Gas to Clv Cols 121 15.0 0 Yes Yes Push ACN until sensors read dry

175) Gas to Clv Cols 121 3.0 0 No Yes Consolidate ACN into vessels


177) Begin Loop 273 0.0 2 No Yes Begin water delivery loop

178) H2O to Clv Cols 126 15.0 0 Yes Yes Deliver water to sensors

179) Gas to Clv Cols 121 15.0 0 Yes Yes Push water until sensors read dry

180) Gas to Clv Cols 121 3.0 0 No Yes Consolidate water into vessels

181) End Loop 274 0.0 0 No Yes End water delivery loop


183) Gas to Clv Cols 121 10.0 0 No Yes Bubble/wash of water/ACN in vessels


185) Sub Label 289 0.0 9 No Yes Drain/wash/dry of coils & blocks

186) Engage All Cols 300 0.0 0 No Yes Force all columns to be active


188) Gas to Coil A 156 15.0 0 No Yes Drain coil A

189) Gas to Coil B 157 15.0 0 No Yes Drain coil B

190) Gas to Coil C 158 15.0 0 No Yes Drain coil C

191) H20 to Coil A 163 8.0 0 No Yes Wash coil A

192) H20 to Coil B 164 8.0 0 No Yes Wash coil B

193) H20 to Coil C 165 8.0 0 No Yes Wash coil C

194) Gas to Coil A 156 15.0 0 No Yes Dry coil A

195) Gas to Coil B 157 15.0 0 No Yes Dry coil B

196) Gas to Coil C 158 15.0 0 No Yes Dry coil C

197) DepUprBlk Rinse 154 3.0 0 No Yes Water rinse for upper depro block

198) DepUprBlk Flush 221 5.0 0 No Yes Flush upper depro block


200) H2O to ClvWaste 130 3.0 0 No Yes Wash lower cleavage blocks w/ water





202) Select Act Cols 283 0.0 0 No Yes Select only cols available for chemistry


204) End of Cycle 272 0.0 0 No Yes Marks end of cycle and all subroutines




The Standard Purification Cycle

Purification CycleSummary

The Purification cycle processes/reactions needed for oligo production are illustrated in the following flow chart (Figure C-3) and the step numbers corresponding to these activities are listed in Table C-5. Table C-6 contains a listing of the standard purification cycle:

Figure C-3 Purification Cycle Flow Chart


Table C-5 Summary of Purification Cycle


1 - 16 Test for Active Columns

� Purification owns the deprotection coils.

� Test for oligos that have ailed leak test in synthesis or cleavage cycles.

� If synthesis or cleavage modules failed leak test, then (a) wait for Syn and Clv modules to complete leak testing, (b) advance the sample collector 3 times for the next set of oligos, (c) drop the coil temperature to the transfer IN setting f(x) 260 and (d) give the depro coils to the cleavage module. Go to Cycle end.

� If synthesis or cleavage modules passed leak test, then close the purification jaw.

17 - 18 Start Here if Columns Active, Leak Test

� Close the purification jaw, begin leak test and check for pass or fail.

19 - 50 Start Purification Cycle or Recover Sample in Coils

� If leak test passes, then go to start of purification cycle at step 51.

� If leak test fails, restore active columns that were originally set from synthesis and execute the recovery of crude oligos from the coils.

� Set ramping function parameters. Wait for deprotection of crude oligos to complete. Drop the coil temperature to the transfer out setting. Set the coils to the transfer in temperature for the next row of oligos.

� Crude A to UV. Clean the purification blocks, transfer line, UV cell and sample collector delivery line. Move the sample collector (SC) rack to the load (close) position for the next sample. Lower the SC needle. Execute the “crude to UV” ramping function and transfer the crude oligo into the SC tube.

� Crude C to UV. Clean the purification blocks, transfer line, UV cell and sample collector delivery line. Move the sample collector (SC) rack to the load (close) position for the next sample. Lower the SC needle Execute the “crude to UV” ramping function and transfer the crude oligo into the SC tube.

� Wash and dry purification blocks, transfer line, UV cell and SC delivery line. Advance the SC for the next sample and open SC (move to front).

� Give cleavage the depro coils and end recovery routine.

51 - 58 Start Here if Purification Passed Leak Test, Wash and Dry Purification Module

� Set regulator 3 to 12 psi. Flush and wash lower pur blocks. Drain and dry pur module. Wash vessels with 2:1 water/ACN. Drain and dry pur module

59 - 60 Test for Crude or Purified Oligos

� Activate only the columns to be purified.

� Check for the columns to be purified. If yes, then go to step 61. If no, then skip to step 105 “Select Act Cols”.


61 - 68 Pre TFA Scrub, ACN/Water Wash of Pur Module

� Two ACN (Go Sub 5) and water (Go Sub 4) washes of the purification module. Drain and dry (Go Sub3) of the purification module.

69 - 70 TFA Scrub of Columns to be Used for Purification

� The OneStep™ columns that are selected to be purified are scrubbed with 3% TFA (Go Sub 9) to remove any product that was not cleaved during the cleavage portion of the cleavage cycle.

71 - 85 Post TFA Scrub, ACN/Water Wash of Pur Module

� Two AN washes of Pur module (Go Sub 5). Split parallel delivery of ACN to Pur Cols and split serial delivery of Water to Pur Cols.

� Loop the split deliveries two times. Water wash of pur module (Go Sub 4). Drain and Dry pur module (Go Sub 3).

86 - 104 Column Prep for Oligos to be Purified

� Split parallel delivery of TEAA to pur sensors. Flush upper pur block. Serial push of TEAA over columns into the vessels. Serial backflush of TEAA from the vessels over the columns out to waste.

� Three loops of a serial backflush to drain and dry the columns. Clean lower; purification block (Go Sub 10).

105 Select All Columns for Transfer of Oligos from Coils into the Purification Vessels

106 -122 Transfer Oligos from the Deprotection Coils into the Purification Vessels

� Set ramping function parameters (Go Sub 11).

� Purification module waits for deprotection to complete.

� Drop the coil temp. to the transfer out temperature.

� Purification block and transfer line are washed before the coil to pur column transfer.

� The coil contents are transferred into the pur vessels in column order C, B, A. The pur block and transfer line are rinsed and dried after the ramping transfer.

123 - 124 Cleavage Owns Depro Coils

� The lower purification is rinsed and dried with water.

� Cleavage owns the deprotection coils.

125 - 126 Test for Crude or Purified Oligos

� Activate only the columns to be purified.

� Check for the columns to be purified. If yes, then go to step 127.

� If no, then skip to step 162 “Select Act Cols”.

127 - 138 Sample Neutralization Loop

� Consolidate the crude sample in the vessels and dry the purification sensors. Flush lower pur block.

� Two parallel sensored deliveries of Acetic Acid to the columns.

Table C-5 Summary of Purification Cycle (continued)



139 - 142 Bonding of Trityl ON Oligos to Columns

� Gently consolidate sample into the purification vessels.

� Start serial backflush over columns at 6 psi, column order A, B, C (Go Sub 7). The serial backflush will ramp the pressure if the purification sensor does not read “dry” at the initial pressure setting of 6 psi.

143 - 148 Cleavage/Detritylation of Bound Oligo from Column

� Wash and dry column with water.

� Start detritylation by delivering 3% TFA to the upper pur sensors. Two loops of (Go Sub 9, TFA push/waits over the column.

149 - 152 Post Cleavage/Detritylation, Wash of Purification Module

� Three loops of water washing of purification module (Go Sub 4), followed by a drain and dry for purification module.

153 - 161 Elution of Oligos from Column into Vessels with 20% ACN

� Parallel split delivery of 0% ACN to Pur Cols that elutes the oligos form the column support into the vessels. Set regulator 3 to 4 psi.

� Gently consolidate and bubble purified oligos in the purification vessels.

162 Select All Columns for Transfer of Oligos from the Purification Vessels to UV Cell




163 - 191 UV Quantitation and Sample Collection

Position A:

� Take UV baseline reading (Go Sub 1).

� Wash the UV cell and SC delivery line (Go Sub 12).

� Advance SC to the back (closed position) for next sample, lower SC needle into tube.

� Consolidate the sample into the vessel. Ramping sensored transfer of oligo, crude or purified, into the UV cell.

� Deliver ACN/water into the pur vessel (Go Sub 14).

� Quantitate oligo and deliver into SC vial (Go Sub 15).

Position B:



� Advance SC to the next position and lower the SC needle into the collection vial.

� Consolidate the sample into the vessel. Ramping sensored transfer of oligo, crude or purified, into the UV ell.


� Quantitate oligo and deliver into SC vial (Go Sub 16).

Position C:



� Advance SC to the next position and lower the SC needle into the collection vial.

� Consolidate the sample into the vessel. Ramping sensored transfer of oligo, crude or purified, into the UV ell.


� Quantitate oligo and deliver into SC vial (Go Sub 17). Wash UC cell and SC delivery line.

� Advance SC to the next position and move the SC to the front (open position).

192 - 199 Post Cycle, Purification Module Wash

� Push ACN/water into the purification vessels until the sensors are dry.

� Wash vessel by bubbling contents. Drain and dry pur module.

� 2:1 water/AN wash of pur vessels (Go Sub 8). Drain and dry purification module (Go Sub 3).




Purification CycleListing

The annotated listing below is provided to aid you in understanding ABI 3948 system cleavage and deprotection:

Table C-6 Standard Purification Cycle (v4.52)

STEP FUNCTION NUM TIME MIS SNS SAFE Comments

1) Begin of Cycle 271 0 0 No Yes Marks beginning of cycle

2) Pur Owns Depro 285 0.0 0 No Yes Purification controls deprotection coils

3) If Any Act Cols 291 0.0 0 No Yes Test for oligos that failed in Syn or Clv

4) Else 280 0.0 0 No Yes If ok, do nothing, else advance SC

5) Wait 259 60.0 0 No Yes Wait for syn/civ module leak testing



8) SC Next 244 0.0 0 No Yes Advance sample collector for bad row

9) SC Next 244 0.0 0 No Yes Advance samPIe collector for bad row

10) SC Next 244 0.0 0 No Yes Advance sample collector for bad row

11) SC Open 245 0.0 0 No Yes Bring sample collector rack to the front

12) Wait Coil Temp 261 0.0 0 No Yes Drop coil temp to transfer OUT temp

13) Set Coil Temp 260 0.0 0 No Yes Set coil temp to transfer IN temp

14) Clv Owns Depro 286 0.0 0 No Yes Gives deprotection coils to cleavage


16) Endif 281 0.0 0 No Yes End of test for bad row

17) Pur Jaw Close 236 0.0 0 No Yes CIose pur jaw, start pur module leak test

18) If Any Act Cols 291 0.0 0 No Yes If test passes, do nothing

19) Else 280 0.0 0 No Yes If test fails, recover crudes from coils

20) Revive Act Cols 319 0.0 0 No Yes Select column that produced crudes

21) Go Sub 287 0.0 11 No Yes Set ramping function parameters

22) Depro Htr Wait 169 0.0 0 No Yes Wait for deDrotection to comDIete



25) Go Sub 287 0.0 2 No Yes CIean blocks and pur transfer line

26) Go Sub 287 0.0 1 No Yes Take UV baseline reading

27) Go Sub 287 0.0 12 No Yes Wash UV cell and SC delivery line

28) SC Close 246 0.0 0 No Yes SC rack to load position for next sample

29) SC Needle Down 248 0.0 0 No Yes Lower needle into collection vial

30) Go Sub A 292 0.0 6 No Yes Recovery: crude coils to UV

31) Xfer UV to SC 215 15.0 0 No Yes Transfer oligo to SC




35) SC Next 244 0.0 0 No Yes Advance SC for next sample

36) Go Sub B 293 0.0 6 No Yes Recovery: crude coils to UV







42) Go Sub C 294 0.0 6 No Yes Recovery: crude coils to UV


44) Go Sub 287 0.0 2 No Yes CIean blocks and our transfer line



47) SC Open 245 0.0 0 No Yes SC rack to front

48) Clv Owns Depro 286 0.0 0 No Yes Cleavage owns the deprotection coils

49) Goto End 290 0.0 0 No Yes End of recovery of crudes routine

50) Endif 281 0.0 0 No Yes To reach here, leak test passed

51) Time Stamp 396 0.0 30 No Yes Time stamp


53) PurLowBlk Flush 218 5.0 0 No Yes Flush lower pur block, may be wet

54) ACN to PurWaste 177 5.0 0 No Yes ACN rinse of lower pur block

55) PurLowBlk Flush 218 5.0 0 No Yes Flush lower pur block

56) Go Sub 287 0.0 3 No Yes Drain & dry vessels/columns/lines


58) Go Sub 287 0.0 3 No Yes Drain & dry,vessels/columns/lines

59) Select Pur Cols 282 0.0 0 No Yes Activate only the columns to be purified

60) If Any Act Cols 291 0.0 0 No Yes Check here for any columns to be purified

61) Go Sub 287 0.0 5 No Yes ACN wash & bubble




65) Go Sub 287 0.0 4 No Yes Water wash & bubble




69) Go Sub 287 0.0 9 No Yes Deliver TFA Scrub and drain

70) Go Sub 287 0.0 9 No Yes Deliver TFA Scrub and drain





75) Begin Loop 273 0.0 2 No Yes Begin ACN/water wash loop

76) ACN to Pur Cols 173 2.2 0 No Yes Start split delivery of ACN

77) ACN to Pur Cols 173 15.0 0 Yes Yes Finish split delivery of ACN to sensor

78) H2O to PurCol A 184 5.0 0 No Yes Water delivery to vessel A

79) H2O to PurCol B 185 5.0 0 No Yes Water delivery to vessel B

Table C-6 Standard Purification Cycle (v4.52) (continued)



80) H2O to PurCol C 186 5.0 0 No Yes Water delivery to vessel C

81) Gas to Pur Cols 203 15.0 0 No Yes Consolidate ACN/water into vessels


83) End Loop 274 0.0 0 No Yes End ACN/water wash loop




87) TEAA to Waste 182 5.0 0 No Yes Prime block w/ TEAA

88) TEAA to PurCols 178 25.0 0 No Yes Start split delivery of TEAA

89) TEAA to PurCols 178 45.0 0 Yes Yes Finish split delivery of TEAA to sensors

90) PurUprBlk Flush 219 2.0 0 No Yes Flush upper pur block

91) Gas to PurCol A 204 10.0 0 No Yes Push TEAA over column into vessel A

92) Gas to PurCol B 205 10.0 0 No Yes Push TEAA over column into vessel B

93) Gas to PurCol C 206 10.0 0 No Yes Push TEAA over column into vessel C


95) Back Flsh Pur A 208 20.0 0 No Yes Drain TEAA from vessel A

96) Back Flsh Pur B 209 20.0 0 No Yes Drain TEAA from vessel B

97) Back Flsh Pur C 210 20.0 0 No Yes Drain TEAA from vessel C

98) Begin Loop 273 0.0 3 No Yes Begin TEAA drain/dry loop

99) Back Flsh Pur A 208 10.0 0 No Yes Drain TEAA from vessel A

100) Back Flsh Pur B 209 10.0 0 No Yes Drain TEAA from vessel B

101) Back Flsh Pur C 210 10.0 0 No Yes Drain TEAA from vessel C

102) End Loop 274 0.0 0 No Yes End TEAA drain/dry loop

103) Go Sub 287 0.0 10 No Yes Clean lower purification block

104) Endif 281 0.0 0 No Yes End of purification column prep

105) Select Act Cols 283 0.0 0 No Yes Select crude and purified columns

106) Go Sub C 294 0.0 2 No Yes Clean blocks and pur transfer line

107) Go Sub 287 0.0 11 No Yes Set ramping function parameters


109) Depro Htr Wait 169 0.0 0 No Yes Wait for deprotection to complete





114) Xfer Coil C 168 12.0 1 Yes No Ramping transfer from coil C to pur sensor

115) Xfer Coil C 168 20.0 0 No No Push sample from sensor to vessel

116) Go Sub B 293 0.0 2 No Yes Clean blocks and pur transfer line

117) Xfer Coil B 167 12.0 1 Yes No Ramping transfer from coil B to pur sensor

118) Xfer Coil B 167 20.0 0 No No Push sample from sensor to vessel

119) Go Sub A 292 0.0 2 No Yes Clean blocks and pur transfer line

120) Xfer Coil A 166 12.0 1 Yes No Ramping transfer from coil A to pur sensor




121) Xfer Coil A 166 20.0 0 No No Push sample from sensor to vessel


123) Clv Owns Depro 286 0.0 0 No Yes Cleavage owns the depro coils


125) Select Pur Cols 282 0.0 0 No Yes Select only the purified oligos

126) If Any Act Cols 291 0.0 0 No Yes Check if there are any oligos to purify


128) Gas to Pur Cols 203 60.0 0 No Yes Extra drying of purification sensors


130) PurLowBlk Flush 218 2.0 0 No Yes Fiush lower pur block

131) Begin Loop 273 0.0 2 No Yes Begin neutralization loop

132) AcAcid to Waste 202 2.0 0 No Yes Prime block with acetic acid

133) AcAcid to Purs 198 3.0 0 No Yes Start split delivery of acetic acid

134) AcAcid to Purs 198 30.0 0 Yes Yes Finish split delivery of acetic acid to sensor

135) Gas to Pur Cols 203 25.0 0 No Yes Mix samples in vessels and dry sensors



138) End Loop 274 0.0 0 No Yes End neutralization loop

139) Gas to Pur Cols 203 15.0 0 No Yes Consolidate samples in vessels

140) Go Sub A 292 0.0 7 No Yes Bind oligo to column A

141) Go Sub B 293 0.0 7 No Yes Bind oligo to column B

142) Go Sub C 294 0.0 7 No Yes Bind oligo to column C

143) Go Sub 287 0.0 4 No No No Water wash & bubble




147) Go Sub 287 0.0 9 No Yes Deliver TFA Detritylation

148) Go Sub 287 0.0 9 No Yes Deliver TFA Detritylation

149) Begin Loop 273 0.0 3 No No Begin water line/column/vessel wash loop

150) Go Sub 287 0.0 4 No No Water wash & bubble


152) End Loop 274 0.0 0 No No End water Erie/column/vessels wash loop

153) 20%ACN to Waste 197 4.0 0 No Yes Prime lower pur block w/ 20% ACN

154) 20%ACN to Purs 193 6.0 0 No Yes Start split delivery of 20% ACN

155) 20%ACN to Purs 193 15.0 0 Yes Yes Finish split deiiverv of 20% ACN, sensor


157) Set Pres Reg 3 303 0.0 4 No Yes Set regulator 3 to 4 psi for elusion


159) Gas to Pur Cols 203 10.0 0 No Yes Elution: push 20% ACN to vessels

160) Endif 281 0.0 0 No Yes End of purification





162) Select Act Cols 283 0.0 0 No Yes Select all crudes and purifieds

163) Go Sub A 292 0.0 1 No Yes Take UV baseline reading

164) Go Sub A 292 0.0 12 No Yes Wash UV cell and SC delivery line

165) SC Close 246 0.0 0 No Yes SC rack to back for next sample

166) SC Needle Down 248 0.0 0 No Yes Lower needle into collection vial

167) Gas to PurCol A 204 7.0 0 No Yes Consolidate sample into vessel A

168) Pur A to UV 211 10.0 3 Yes Yes Ramping delivery of A to UV sensor

169) Pur A to UV 211 20.0 0 No Yes Finish delivery of A to UV cell

170) Go Sub A 292 0.0 14 No Yes Deliver ACN/water to vessels

171) Go Sub A 292 0.0 15 No Yes Quantitate & collect for sample A

172) Go Sub B 293 0.0 1 No Yes Take UV baseline reading

173) Go Sub B 293 0.0 12 No Yes Wash UV cell and SC delivery line


175) Gas to PurCol B 205 7.0 0 No Yes Consolidate sample into vessel B

176) Pur B to UV 212 10.0 3 Yes Yes Ramping delivery of B to UV sensor

177) Pur B to UV 212 20.0 0 No Yes Finish delivery of B to UV cell

178) Go Sub B 293 0.0 14 No Yes Deliver ACN/water to vessels

179) Go Sub B 293 0.0 16 No Yes Quantitate & collect for sample B

180) Go Sub C 294 0.0 1 No Yes UV baseline reading

181) Go Sub C 294 0.0 12 No Yes Wash UV cell and SC delivery line


183) Gas to PurCol C 206 7.0 0 No Yes Consolidate sample into vessel C

184) Pur C to UV 213 10.0 3 Yes Yes Ramping delivery of C to UV sensor

185) Pur C to UV 213 20.0 0 No Yes Finish delivery of C to UV cell

186) Go Sub C 294 0.0 14 No Yes Deliver ACN/water to vessels

187) Go Sub C 294 0.0 17 No Yes Quantitate & collect for sample C



190) SC Open 245 0.0 0 No Yes SC rack to front



193) Gas to Pur Cols 203 45.0 0 Yes Yes Push contents into vessels until sensor dry

194) Gas to Pur Cols 203 10.0 0 No Yes Bubble contents in vessels






200) Sub Label 289 0.0 1 No Yes Take UV baseline reading


202) SC Waste 242 0.0 0 No Yes Send the SC to the waste




203) H2O to PurWaste 187 5.0 0 No Yes Water rinse for lower purification block

204) H2O to UV 214 5.0 0 No Yes Water rinse of UV cell

205) SC Block Flush 222 5.0 0 No Yes Flush lower pur blocks and UV cell dry

206) Wait 259 10.0 0 No Yes Wait/soak

207) UV Reading 216 2.0 0 No Yes Take baseline UV reading (misc=0)

208) Flsh UV toWaste 217 6.0 0 No Yes Flush UV cell dry



211) Xfer UV to SC 215 10.0 0 No Yes Flush UV cell to SC waste


213) Sub Label 289 0.0 2 No Yes Clean blocks and pur transfer line





218) Pur Xfer Rinse 171 2.0 0 No Yes Water rinse pur transfer line

219) Pur Xfer Flush 172 15.0 0 No Yes Flush of pur transfer line

220) Set Pres Reg 1 301 0.0 8 No Yes Pur to UV starting pressure

221) DepLowBlk Flush 153 2.0 0 No No Flush lower depro block

222) Return Sub 288 0.0 0 No No End of subroutine 2.

223) Sub Label 289 0.0 3 No Yes Drain & dry vessels/columns/lines


225) Back Flush Purs 207 6.0 0 No Yes Wet sensors for all active vessels

226) Back Flush Purs 207 45.0 0 Yes Yes Drain all vessels until sensors read dry

227) Back Flsh Pur A 208 6.0 0 No Yes Complete drain of vessel A

228) Back Flsh Pur B 209 6.0 0 No Yes Complete drain of vessel B

229) Back Flsh Pur C 210 6.0 0 No Yes Complete drain of vessel C

230) Begin Loop 273 0.0 2 No Yes Begin vessel drain/dry loop

231) Gas to Pur Cols 203 1.0 0 No Yes Reverse drying -

232) Back Flush Purs 207 4.0 0 No Yes Backflush drying

233) End Loop 274 0.0 0 No Yes End vessel drain/dry loop


235) Sub Label 289 0.0 4 No Yes Water wash & bubble


237) H2O to Pur Cols 183 3.0 0 No Yes Start split delivery of water

238) H2O to Pur Cols 183 15.0 0 Yes Yes Finish split delivery of water to sensor

239) Gas to Pur Cols 203 15.0 0 Yes Yes Push water to vessel until sensor dry

240) Gas to Pur Cols 203 6.0 0 No Yes Bubble/wash purification columns


242) Sub Label 289 0.0 5 No Yes ACN wash & bubble





244) ACN to Pur Cols 173 4.0 0 No Yes Start split delivery of ACN

245) ACN to Pur Cols 173 15.0 0 Yes Yes Finish split delivery of ACN to sensor

246) Gas to Pur Cols 203 15.0 0 Yes Yes Push ACN to vessel until sensor dry

247) Gas to Pur Cols 203 6.0 0 No Yes Bubble/wash purification columns


249) Sub Label 289 0.0 6 No Yes Recovery: crude coils to UV

250) Crude A to UV 135 12.0 1 Yes Yes Transfer coils contents to UV sensor

251) Crude A to UV 135 20.0 0 No Yes Push coils contents into cuvette

252) Crude B to UV 136 12.0 1 Yes Yes Transfer coil B contents to UV sensor

253) Crude B to UV 136 20.0 0 No Yes Push coil B contents into cuvette

254) Crude C to UV 137 12.0 1 Yes Yes Transfer coil C contents to UV sensor

255) Crude C to UV 137 20.0 0 No Yes Push coil C contents into cuvette


257) Sub Label 289 0.0 7 No Yes Bind oligo to column A/B/C

258) Set Pres Reg 3 303 0.0 12 No Yes Set regulator 3 to 12 psi .




262) Back Flush Purs 207 15.0 0 No Yes Wet sensors for all active vessels

263) Back Flush Purs 207 45.0 3 Yes Yes Bind vessel contents until sensor read dry


265) Back Flush Purs 207 15.0 0 No Yes Complete vessel drain


267) Sub Label 289 0.0 8 No Yes 2:1 water/ACN vessel wash


269) ACN to Pur Cols 173 4.0 0 No Yes Start split delivery of ACN to columns

270) ACN to Pur Cols 173 15.0 0 Yes Yes Split delivery of ACN to sensors

271) Gas to Pur Cols 203 15.0 0 Yes Yes Push ACN to vessels until sensor reads dry

272) Gas to Pur Cols 203 3.0 0 No Yes Bubble contents in vessel


274) Begin Loop 273 0.0 2 No Yes Begin water wash loop

275) H2O to Pur Cols 183 4.0 0 No Yes Start split delivery of water to column

276) H2O to Pur Cols 183 15.0 0 Yes Yes Finish split delivery of water to sensor

277) Gas to Pur Cols 203 15.0 0 Yes Yes Push water to vessel until sensor read dry

278) Gas to Pur Cols 203 3.0 0 No Yes Bubble contents in vessel

279) End Loop 274 0.0 0 No Yes End water wash loop


281) Gas to Pur Cols 203 8.0 0 No Yes Bubble water/ACN in vessels


283) Sub Label 289 0.0 9 No Yes TFA detritylation/scrub and drain





285) PurLowBlk Flush 218 2.0 0 No Yes Flush lower purification block

286) TFA to Pur Cols 188 6.0 0 No Yes Start split delivery of TFA to columns

287) TFA to Pur Cols 188 25.0 0 Yes Yes Finish split delivery of TFA to sensors

288) Wait 259 15.0 0 No Yes Initial detritylation/scrub wait

289) Begin Loop 273 0.0 2 No Yes Begin detritylaylon/scrub loop

290) TFA to PurCol A 189 1.0 999 No Yes Push fresh TFA over column A or wait

291) TFA to PurCol B 190 1.0 999 No Yes Push fresh TFA over column B or wait

292) TFA to PurCol C 191 1.0 999 No Yes Push fresh TFA over column C or wait

293) Wait 259 15.0 0 No Yes Wait for detritylation

294) End Loop 274 0.0 0 No Yes End detritylayion/scrub loop

295) Begin Loop 273 0.0 6 No Yes Begin detritylaylon/scrub loop

296) Gas to PurCol A 204 0.6 0 No Yes Gas push of fresh TFA over column A

297) Gas to PurCol B 205 0.6 0 No Yes Gas push of fresh TFA over column B

298) Gas to PurCol C 206 0.6 0 No Yes Gas push of fresh TFA over column C

299) Pur Vent 220 2.0 0 No Yes Vent to allow TFA push

300) Wait 259 23.0 0 No Yes Wait for detritylation

301) End Loop 274 0.0 0 No Yes End detritylaylon/scrub loop


303) Tggle Vlves On 262 0.0 70 No Yes Select lower pur block to halog waste

304) Back Flsh Pur A 208 12.0 0 No Yes Drain vessel A to halo waste

305) Back Flsh Pur B 209 12.0 0 No Yes Drain vessel B to halo waste

306) Back Flsh Pur C 210 12.0 0 No Yes Drain vessel C to halo waste


308) Back Flsh Pur A 208 10.0 0 No Yes Dry line/column/vessel A

309) Back Flsh Pur B 209 10.0 0 No Yes Dry line/column/vessel B

310) Back Flsh Pur C 210 10.0 0 No Yes Dry line/column/vessel C

311) Gas to Pur Cols 203 2.0 0 No Yes Parallel drv in reverse direction

312) Back Flush Purs 207 8.0 0 No Yes Finish parallel dry line/column/vessel

313) Tggle Vlves Off 263 0.0 70 No Yes Deselect lower pur block to halog waste


315) Sub Label 289 0.0 10 No Yes Clean lower purification block

316) H2O to PurWaste 187 1.0 0 No Yes Water rinse of lower pur block


318) PurLowBlk Flush 218 6.0 0 No Yes Rinse lower pur block


320) Sub Label 289 0.0 11 No Yes Set ramping function parameters

321) Set Rmp Fxn Sns 265 0.0 10 No Yes Set ramp parms: # of total readings

322) Set Rmp Fxn Trp 266 0.0 5 No Yes Set ramp parms: # of readings to trip

323) Set Rmp Fxn Chk 267 0.0 5 No Yes Set ramp parms: # of readings to verify

324) Set Rmp Fxn Dly 268 0.0 60 No Yes Set ramp parms: # of ms to delay verify





326) Sub Label 289 0.0 12 No Yes Wash UV cell and SC delivery line


328) SC Waste 242 0.0 0 No Yes Send SC to waste position

329) H2O to PurWaste 187 3.0 0 No Yes Water rinse of lower pur block








337) Sub Label 289 0.0 13 No Yes Not used


339) Sub Label 289 0.0 14 No Yes Deliver ACN/water to vessels

340) ACN to Pur Cols 173 4.0 0 No Yes Start split delivery of ACN to vessels

341) ACN to Pur Cols 173 15.0 0 Yes Yes Finish split delivery of ACN to sensors

342) H2O to Pur Cols 183 10.0 0 No Yes Parallel delivery of water to vessels






348) Sub Label 289 0.0 15 No Yes Quantitate & collect for sample A

349) UV Reading 216 2.0 1 No Yes Take UV reading (misc of 1 = A)

350) Xfer UV to SC 215 15.0 0 No Yes Transfer oligos to SC


352) Sub Label 289 0.0 16 No Yes Quantitate & collect for Sample B

353) UV Reading 216 2.0 2 No Yes Take UV reading (misc of 2 = B)



356) Sub Label 289 0.0 17 No Yes Quantitate & collect for sample C

357) UV Reading 216 2.0 3 No Yes Take UV reading (misc of 3 = C)



360) End of Cycle 272 0.0 0 No Yes Marks end of cycle and all subroutines




Procedures

General Procedures are similar to cycles in that they consist of a set of functions that perform an operation. Procedures, if performed during a run, are typically performed once, while cycles are repeatedly performed during a run.

Edit BeginProcedure View

The ABI 3948 System is provided with one Begin procedure. The Start Up procedure primes all reagent delivery lines, from the reservoir to the reagent valve block, with fresh reagent.

IMPORTANT Always use this procedure prior to beginning a synthesis. Failure to perform the Begin procedure can cause a synthesis failure.

The Start Up Procedure

An example of this procedure is listed in Table C-7. To modify the procedure, copy it into a blank Begin procedure in the Edit Begin Procedure view (see “Edit Begin Procedure View” on page 6-20).

Table C-7 The Start Up Procedure (v1.35 shown)

STEP FUNCTION NUM TIME MISC SNS SAFE

1) Begin of Cycle 271 0.0 0 No Yes

2) Send Message 269 0.0 5 No Yes

3) DB 4.20H, 2.20H 110 0.0 0 No Yes

4) Clv Owns Depro 286 0.0 0 No Yes

2) Send Message 269 0.0 5 No Yes

3) DB 4.20H, 2.20H 110 0.0 0 No Yes

4) Clv Owns Depro 286 0.0 0 No Yes

5) Check Snsr Cal 320 0.0 0 No Yes

6) Heater Off 258 0.0 0 No Yes

7) Pres Reg AllOff 315 0.0 0 No Yes

8) Pres Reg AllOn 314 0.0 0 No Yes

9) Set Pres Reg 1 301 0.0 12 No Yes










19) Pres Reg AllOn 314 0.0 0 No Yes

20) Wait 259 15.0 0 No Yes

21) SynLowBlk Flush 65 5.0 0 No Yes

22) Go Sub 287 0.0 1 No Yes

23) IODINE to Waste 37 2.0 0 No Yes


24) Go Sub 287 0.0 1 No Yes

25) TCA to Trityl 42 4.0 0 No Yes

26) ACN to Trityl 108 2.0 0 No Yes

27) LowTrityl Flush 106 5.0 0 No Yes

28) AC2O to Waste 31 2.0 0 No Yes

29) Go Sub 287 0.0 1 No Yes

30) NMI to Waste 32 2.0 0 No Yes

31) Go Sub 287 0.0 1 No Yes

32) T AMIDITE Waste 77 3.0 0 No Yes

33) C AMIDITE Waste 76 3.0 0 No Yes

34) G AMIDITE Waste 75 3.0 0 No Yes

35) A AMIDITE Waste 74 3.0 0 No Yes

36) 8 AMIDITE Waste 81 3.0 0 No Yes




40) Go Sub 287 0.0 1 No Yes

41) TET to Waste 52 2.0 0 No Yes

42) Go Sub 287 0.0 1 No Yes

43) Clv Blk Flush 144 10.0 0 No Yes

44) H2O to ClvWaste 130 5.0 0 No Yes


46) Ammonia toWaste 115 3.0 0 No Yes

47) Dep Xfer Rinse 151 3.0 0 No Yes


49) ACN to ClvWaste 120 2.0 0 No Yes

50) DepLowBlk Flush 153 5.0 0 No Yes

51) Gas to Coil A 156 15.0 0 No Yes

52) Gas to Coil B 157 15.0 0 No Yes

53) Gas to Coil C 158 15.0 0 No Yes

54) Pur Xfer Rinse 171 3.0 0 No Yes

55) Pur Xfer Flush 172 10.0 0 No Yes

56) Go Sub 287 0.0 2 No Yes

57) 20%ACN to Waste 197 2.0 0 No Yes

58) Go Sub 287 0.0 2 No Yes

59) H2O to PurWaste 187 2.0 0 No Yes

60) Go Sub 287 0.0 2 No Yes

61) AcAcid to Waste 202 3.0 0 No Yes

62) Go Sub 287 0.0 2 No Yes

63) TFA to Waste 192 2.0 0 No Yes

64) Tggle Vlves On 262 0.0 70 No Yes

Table C-7 The Start Up Procedure (v1.35 shown) (continued)



Edit End ProcedureView

Use the End procedure to flush instrument reagent lines and valve blocks at the end of each synthesis run. An example of the standard end procedure provided with the instrument is listed in Table C-8. To modify this procedure, copy it into a blank End procedure (see “Edit End Procedure View” on page 6-21).

65) PurLowBlk Flush 218 2.0 0 No Yes

66) ACN to PurWaste 177 2.0 0 No Yes


68) Tggle Vlves Off 263 0.0 70 No Yes

69) TEAA to Waste 182 5.0 0 No Yes




73) Go Sub 287 0.0 2 No Yes

74) SC Waste 242 0.0 0 No Yes

75) H2O to UV 214 10.0 0 No Yes

76) Flsh UV toWaste 217 10.0 0 No Yes

77) Xfer UV to SC 215 5.0 0 No Yes

78) SC Home 243 0.0 0 No Yes

79) SC Open 245 0.0 0 No Yes

80) Goto End 290 0.0 0 No Yes

81) Sub Label 289 0.0 1 No Yes

82) ACN to SynWaste 47 3.0 0 No Yes

83) SynLowBlk Flush 65 6.0 0 No Yes

84) Return Sub 288 0.0 0 No Yes

85) Sub Label 289 0.0 2 No Yes



88) Return Sub 288 0.0 0 No Yes

89) End of Cycle 272 0.0 0 No Yes

Table C-7 The Start Up Procedure (v1.35 shown) (continued)


Table C-8 End Procedure (v1.11 shown)


1 Begin of Cycle 271 0.0 0 No Yes

2 Heater Off 258 0.0 0 No Yes

3 Set Pres Reg 1 301 0.0 12 No Yes


5 PurUprBlk Flush 219 20.0 0 No Yes

6 PurLowBlk Flush 218 20.0 0 No Yes

7 Tggle Vlves On 262 0.0 70 No Yes


9 Tggle Vlves Off 263 0.0 70 No Yes


10 DepUprBlk Flush 221 20.0 0 No Yes


12 DepLowBlk Flush 153 20.0 0 No Yes

13 Clv Blk Flush 144 20.0 0 No Yes



16 AMIDITE Vent 101 10.0 0 No Yes

17 ACN to SynWaste 47 10.0 0 No Yes

18 SynLowBlk Flush 65 20.0 0 No Yes

19 LowTrityl Flush 106 20.0 0 No Yes

20 Mix AMIDITE T 93 3.0 0 No Yes

21 Mix AMIDITE C 92 3.0 0 No Yes

22 Mix AMIDITE G 91 3.0 0 No Yes

23 Mix AMIDITE A 90 3.0 0 No Yes

24 Mix AMIDITE 8 97 3.0 0 No Yes





29 SynUprBlk Flush 105 20.0 0 No Yes




33 Dep Xfer Flush 152 5.0 0 No Yes

34 Dep Xfer Rinse 151 3.0 0 No Yes

35 Dep Xfer Flush 152 5.0 0 No Yes

36 Gas to Coil A 156 15.0 0 No Yes

37 Gas to Coil B 157 15.0 0 No Yes

38 Gas to Coil C 158 15.0 0 No Yes

39 H20 to Coil A 163 8.0 0 No Yes

40 H20 to Coil B 164 8.0 0 No Yes

41 H20 to Coil C 165 8.0 0 No Yes

42 Gas to Coil A 156 15.0 0 No Yes

43 Gas to Coil B 157 15.0 0 No Yes

44 Gas to Coil C 158 15.0 0 No Yes

45 Pur Xfer Flush 172 10.0 0 No Yes

46 Pur Xfer Rinse 171 5.0 0 No Yes


48 Open All Jaws 237 0.0 0 No Yes

49 Send Message 269 0.0 5 No Yes

50 End of Cycle 272 0.0 0 No Yes

Table C-8 End Procedure (v1.11 shown) (continued)



Edit BottleProcedures View

Two types of procedures are located on the Edit Bottle Procedures view: Autodilute and Change procedures.

Phosphoramidite Autodilution

Use one of the Autodilute procedures to automatically dilute powdered β-cyanoethyl phosphoramidites in 2.0 gram bottles. The table below lists the Autodilute procedures available and the purpose of each.

IMPORTANT During this procedure, back-flushed phosphoramidite and excess acetonitrile contaminate the bottle contents. If you want to remove and save the phosphoramidites for later use, do not initiate this procedure.

Bottle Change Procedures (Manual Dilution)

Change Base (Chg base) Procedures for Positions A through G and Positions 5 through 8 on the instrument are intended for phosphoramidites manually diluted by the user. After you have manually diluted a bottle of phosphoramidite, use the change procedure for the phosphoramidite position on which you want to install the new bottle.

The change procedure provided in Table C-10 is representative of the Chg procedures provided. Besides change procedures for the four phosphoramidite positions, procedures are provided for changing tetrazole and ammonia.

Table C-9 List of Phosphoramidite Autodilution Procedures

Name Purpose

Autodil AGCT x.xx Simultaneously autodilutes phosphoramidites at Positions A, G, C, and T.

Autodilute-A x.xx Autodilutes phosphoramidite at Position A.

Autodilute-G x.xx Autodilutes phosphoramidite at Position G.

Autodilute-C x.xx Autodilutes phosphoramidite at Position C.

Autodilute-T x.xx Autodilutes phosphoramidite at Position T.

Table C-10 Bottle Change Procedure

Step Function Name Number Time Misc SNS Safe


2 Set Press Reg 9 309 0.0 0 No Yes

3 AMIDITE Vent 101 30.0 0 No Yes

4 ACN AMIDITE 5 86 4.0 0 No Yes

5 Mix ACN AMIDITE 5 94 5.0 0 No Yes



8 Pause Chemistry 291 0.0 0 No Yes

9 Set Press Reg 9 309 0.0 6 No Yes

10 ACN to SynWaste 47 3.0 0 No Yes


MiscellaneousProcedures

The Miscellaneous Procedures view of the 3948Control software application contains the following:

� A procedure for setting system pressures

� Procedures for calibrating the sensors and verifying calibration

� A procedure for priming amidite positions

� Procedures for cleaning columns and lines

� A procedure for emptying ACN lines

� Procedures for testing hardware

� Procedures for testing the pressure module and sample collector

� A Janitor procedure to clean up the system before shutdown

11 SynLowBlk Flush 65 3.0 0 No Yes


Table C-10 Bottle Change Procedure (continued)

Step Function Name Number Time Misc SNS Safe


System Messages

Instrument Statusand Message

Indicators

Instrument Status Messages

The following instrument status messages appear in the upper right corner of all Synthesizer Window views to indicate the current instrument condition:

� Ready - the instrument is idle and ready for a run to be initiated

� Running - the instrument is currently running chemistry

� Interrupted - a run in progress has been interrupted

� Manual Control - a manual control action is in progress

Note It is important to be aware of what state the instrument is in at the time of a system message.

Message Waiting/Critical Message Waiting

These messages are presented in the upper left corner of all Synthesizer Window views except the Monitor Chemistry View, to indicate that an important message is currently presented in the log on the bottom of the Monitor Chemistry View.

Types of Messages There are three types of messages generated for the 3948 instrument and flagged by message indicators:

� Regular Reports to the Monitor Chemistry view

� Critical messages presented in the Monitor Chemistry window

� Microphone Only messages or messages stored in a Microphone file (not shown in Monitor Chemistry).

These message types are flagged as follows:

� Regular Reports messages are flagged on any ABI 3948 System Control view by “Message Waiting”.

� Critical messages are flagged on any ABI 3948 System Control view by “Critical Message Waiting.”

� Microphone Only messages are not available during a run but can be examined at the conclusion of the run.

Proceed to the log on the bottom of the Monitor Chemistry view to see the particular message indicated by a message indicator.


Categories ofMessages

Messages presented in the Monitor Chemistry view or in a Microphone file can be further categorized as Operator, Instrument, or General Activity messages:

� Operator messages are the messages prefaced by “Error:” or “Caution:”

� Instrument messages are the messages prefaced by “Attention:” or “Note”.

� General Activity messages are the messages without the “Error”, “Caution”, “Attention”, or “Note” prefaces.

Some examples of conditions which may generate these three categories of messages are listed under the discussions for the message types below. For a listing of the relative severity of Operator and Instrument message, see “Hierarchical Messages” on page C-43.

MessageConventions

The type of activities which may be flagged by the three categories of message are listed below:

Operator Messages

Operator messages, those messages prefaced by “Error” or “Caution,” are generated by the types of activities outlined below.

Note A hierarchy of Operator messages is presented under “Hierarchical Messages” on page C-43.

Message Preface Type of Activity

Error Characterized by Cycle programming or Manual Control parameter errors:

– Using functions from the wrong chemistry module controller

– Ramping function parameter(s) not set or out of range

– Other out-of-range values (illegal pressure or regulator number)

Note Messages report values substituted at range limits.

Caution Characterized by Discretionary bending of optimal operational rules:

– Allowing deliveries to open jaws

– Setting temperature wait time too short to reach setpoint


Instrument Messages

Instrument messages, those messages prefaced by “Attention” or “Note,” are generated by the types of activities outlined below.

General Activity Messages

General Activity messages, messages with no preface, are generated by the types of activities outlined below.

The three types of messages are listed in separate subsections below with the information needed to interpret them.

Note The examples provided in the three listings are representative of those you might see in the Monitor Chemistry view window or in the Microphone file for a run and are generally self explanatory.

Regular ReportsMessages

This type of message contains all three categories of messages: Operator, Instrument, and General Activity.

Messages from these functions: Send Message (269) and Comment (270)

Note The Send Message command is used in bottle change procedures to instruct the operator to take some action.

Resetting the Database.Pausing system. Resuming System. Pausing system: will auto-resume in 15 minutes.Data Base has been reset.


Attention Reports events likely to impact instrument performance

– - Transfers missed

– - Sensor never triggered

– - Jaw leak test failures and subsequent oligo drop outs

Note Indicates minor and/or infrequent operational events:

– Retries

– Recoveries

– Sensor did not verify

– Other miscellaneous events


No leader or message preface

Reports instrument actions and hardware status, mostly to Microphone:

– Run and Manual Control start/end/abort/pause/resume

– Turntable moves

– Jaw activity: open/close/leak test results

– Regulator setpoint changes

– Other: low pressure, a/d reference voltage, s/c motor problems

– Etc.


Changed Instrument Name. Pause Switch Pushed.Resume with columns 4-6 in the load position.Starting Chemistry Run: [Image version], [Database version].Aborting Chemistry: [Image version], [Database version].Chemistry Done: [Image version], [Database version].Stopping Manual Control Action. Aborting Manual Control. Turntable move 10 completed.Pausing System at turntable position 12.Jaw pressure check retry failed during synthesis.Initial synthesis pressure check failed: now retrying.Regulator '23' does not exist.Power Fail at 10/27/97 09:23:36.A to D reference voltage 3.5 out of range.Rack selection changed.Note: turntable move retried.Note: low input pressure detected.Note: used Xfer Clv Col A recovery.Note: function BackFlsh Purs retried 4 times.(to µphone only if <4)Note: function TCA to Syn Cols retried A column 4 times.Note: heater was turned off after 180 minutes.Attention: Xfer Clv Col C transfer missed.Attention: C+TET to column B sensor never triggered.Attention: sensor never triggered at pur step 188.Attention: leak detected in synthesis jaw/blocks.Attention: oligos 10-12 discontinued during synthesis.Caution: temp set to 35°, reached 36° in 300 seconds.Error: cannot set regulator 10 to 15 psi, 12 psi limit used.Error: used minimum heater setpoint of 15 instead of 10.Error: used maximum heater setpoint of 70 instead of 85.Error 0 readings requested: minimum valule of 1 used.Error: 10 trip readings requested, used current max. 7.Error: 10 check readings requested, used current max. 5.Error: 20 readings requested: maximum value of 10 used.Error: 10 trip readings reduced to new maximum of 7.sError: 10 check readings reduced to new maximum of 7.Error: 20000ms sensor delay requested: maximum 2280.Error: misc parameter must be: 1 = syn, 2 = clv, or 3 = pur.Error: If First Cycle only works in synthesis cycles.Error: End Row SCP/123 only works in Manual Control.Error: no active oligos in cleavage to be discontinued.Error: cannot end row because purification is inactive.Error: oligos 7-9 now in cleavage are already discontinued.Error: 999 heater time requested, used maximum 480.


Critical Messages This type of message contains only the General Activity category of message.

Note Send Message function messages maybe be made 'critical' by selecting liquid sensor use.

Manual Control Paused: Resume instead of Running. Sample Collector door open. Pausing System. Jaw move failure. Pausing System. Jaws open? Check jaws and Instrument Monitor Window.Invalid RunS file: no cycles selected to run.Invalid SeqL file: maximum sequence length exceeded. Turntable May Be Out Of Position.Regulators shut down due to low input pressure. Synthesis jaw did not open. Cleavage jaw did not close. Sensor Calibration Problem. Pausing System. Illegal valve set! Valves left off. Pausing System.TTextFile::WriteData index = 501, max = 500TTextFile::ReadData index = 501, max = 500TStructFile::WriteData index = 501, max = 500TStructFile::ReadData index = 501, max = 500Jaws open: clv & pur! Valves left off. Pausing System.Powerfail interrupted jaw open. Pausing System.Powerfail interrupted jaw close. Pausing System.Powerfail interrupted needle up move Pausing System.Powerfail interrupted needle down move Pausing System.Powerfail interrupted turntable move Pausing System.Coil temperature 100°C is out of range. Pausing System.Sample Collector Z Motor Timed Out. Pausing System.Instrument in use: rack selection not changed.Row ended: advance collector into position for next row.Caution: reagent deliveries to open jaws enabled. Error: Amidite bottle 'A' appears to be empty.

Microphone-onlymessages

This type of message contains only the General Activity category of message.

Starting Manual Control Action. Pausing Manual Control. Resuming Manual Control. Manual Control Action Complete. Coil temperature has been set to 35°C.Coil temperature has been set to 65°C for deprotection.Temp set to 38°, reached 38° in 281 seconds.Name or Msg;Snsr; msecs; °C;Step; Ttbl(Microphone log column headings)Synthesis A cycle ended.Cleavage cycle ended.Purification cycle ended.Changing regulator 10 from 6 psi to 12 psi.LogSensor Output (the standard sensor function report line)Wet(11B) ≥ 9/10 Hg: 2232 1 1 1 1 1 1 1 1 1 5 14|14 (histogram)Voltage[A]: -2.13Results[A]: 3.2 odu 18.3 pmole


Syn P 0.01 N -.-- Clv P 0.02 N-.-- Pur F 0.54 P 0.49 (1st line of 2)Tested at 12 psi, 2.00 passes (2nd line of 2)Ramping function parameter not set. Pausing System.PSIs: 10.200 6.532 10.200 6.532 10.200 6.532 10.200 6.532 10.200 6.532 Leak Test: 10.02 9.98 0.04 PWait On Pur Flg; 00:12:21Depro Heater/Fan Test STARTINGDepro Heater Test RUNNING Temp= 27, HtrCur=1200Depro Fan Test RUNNING Temp=35, FanCur= 60Depro Heater/Fan Test COMPLETEDepro Htr Wait ensuring 60 minute deprotection; 00:12:21Time Stamp; 00:12:21; 33Coils have been hot for 63 minutes.Synthesis jaw open elapsed time = 1567 ms.Cleavage jaw close elapsed time = 1786 ms.Sample Collector Z Motor Home Flag Error.Note: function BackFlsh Purs retried 1 times.Note: sensor 23B did not verify.Note: function TCA to Syn Cols retried A column 3 times.

HierarchicalMessages

The list below presents a hierarchical list of Operator and Instrument category messages, ranging from conditions most likely to negatively affect a run at the top to those least likely at the bottom. This hierarchy applies both to the individual messages within each type (Error, Caution, Attention, Note) and to the relative severity of each of these types of message.

Error: Amidite bottle 'G' appears to be empty.B+TETError: 0 readings requested, used minimum value 1.RampingError: 20 readings requested, used maximum value 10.Error: 10 trip readings reduced to new maximum of 7.Error: 9 check readings reduced to new maximum of 7.Error: 0 trip readings requested, used minimum value 1. Error: 10 trip readings requested, used current max. 7. Error: 9 check readings requested, used current max. 7. Error: 20000ms sensor delay requested, used maximum 2280.Error: used minimum heater setpoint of 15 instead of 10.HeaterError: used maximum heater setpoint of 70 instead of 85.Error: cannot set regulator 10 to 15 psi, 12 psi limit used.OtherError: misc parameter must be: 1 = syn, 2 = clv, or 3 = pur.Error: If First Cycle only works in synthesis cycles.Error: End Row SCP/123 only works in Manual Control.Error: no active oligos in cleavage to be discontinued.Error: oligos 7-9 now in cleavage are already discontinued.Error: 999 heater time requested, used maximum 480.

Caution: reagent deliveries to open jaws enabled.Caution: temp set to 35°, reached 36° in 300 secs.

Attention: 'G'+TET to column B sensor never triggered. Attention: sensor never triggered at pur step 10. Attention: Xfer Clv Col C transfer missed.Attention: Leak detected during in synthesis jaw/blocks.Attention: Oligos 10-12 discontinued during synthesis.


Note: function BackFlsh Purs retried 1 times.Note: function TCA to Syn Cols retried A column 3 times.Note: sensor 23B did not verify.Note: used Xfer Clv Col A recovery.Note: turntable move retried. Note: low input pressure detected.Note: heater was turned off after 180 minutes.

ABI 3948 System Function List and Other Cycles D-1

3948 System Function List and Other Cycles D

In This Appendix

Topics Covered This appendix contains a detailed ABI 3948 System function list as well as the additional purification cycles listed below:

Topic See page

ABI 3848 System Function List D-2

The Purification Dye Cycle D-33

The Purification Biotin Cycle D-42

D

D-2 ABI 3948 System Function List and Other Cycles

ABI 3948 System Function List

List of FunctionsTable D-1 lists the functions available for the ABI 3948 System.

Table D-1 ABI 3948 System Function List (v4.20, 2.20)

Numb Name Valve List Valve Description

1 B+ TET to Syns 4 Tetrazole

22 SynUpr to Waste

Note Function 1 is a “macro” function. This means that the particular valves used vary depending upon the sequence composition and the column to which delivery is made. This function makes three separate serial amidite deliveries to one column rather than a single simultaneous (parallel) delivery to all three columns

2 A + TET to Syn A 4 Tetrazole

9 "A" Amidite

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper

3 G+ TET to Syn A 4 Tetrazole

10 "G" Amidite

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper

4 C+ TET to Syn A 4 Tetrazole

11 "C" Amidite

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper

5 T+ TET to Syn A 4 Tetrazole

12 "T" Amidite

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper

6 5+ TET to Syn A 4 Tetrazole

5 "5" Amidite

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper


6 "6" Amidite

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper



7 "7" Amidite

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper


8 "8" Amidit

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper

10 A+ TET to Syn B 4 Tetrazole

9 "A" Amidite

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper

11 G+ TET to Syn B 4 Tetrazole

10 "G" Amidite

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper

12 C+ TET to Syn B 4 Tetrazole

11 "C" Amidite

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper

13 T+ TET to Syn B 4 Tetrazole

12 "T" Amidite

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper

14 5+ TET to Syn B 4 Tetrazole

5 "5" Amidite

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper


6 "6" Amidite

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper





7 "7" Amidite

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper


8 "8" Amidite

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper

18 A+ TET to Syn C 4 Tetrazole

9 "A" Amidite

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper

19 G+ TET to SynC 4 Tetrazole

10 "G" Amidite

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper

20 C+ TET to Syn C 4 Tetrazole

11 "C" Amidite

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper

21 T+ TET to Syn C 4 Tetrazole

12 "T" Amidite

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper

22 5+ TET to Syn C 4 Tetrazole

5 "5" Amidite

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper


6 "6" Amidite

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper


7 "7" Amidite




13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper


8 "8" Amidite

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper

26 Coupling Wait Waits according to the time in the B+TET Calibration table.

27 CAP to Syn Cols 13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

16 Xfr to SynLow

17 NMI

18 Ac20

22 SynUpr to Waste

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

28 CAP to SynCol A 15 Syn Col A Lower

16 Xfr to SynLow

17 NMI

18 Ac20

22 SynUpr to Waste

26 Syn Col A Upper

29 CAP to SynCol B 14 Syn Col B Lower

16 Xfr to SynLow

17 NMI

18 Ac20

22 SynUpr to Waste

25 Syn Col B Upper

30 CAP to SynCol C 13 Syn Col C Lower

16 Xfr to SynLow

17 NMI

18 Ac20

22 SynUpr to Waste

24 Syn Col C Upper

31 AC20 to Waste 18 Ac20

21 SynLow to Waste

32 NMI to Waste 17 NMI




21 SynLow to Waste

33 IODINE to Syns 13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

16 Xfr to SynLow

20 Iodine

22 SynUpr to Waste

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

34 IODINE to Syn A 15 Syn Col A Lower

16 Xfr to SynLow

20 Iodine

22 SynUpr to Waste

26 Syn Col A Upper

35 IODINE to Syn B 14 Syn Col B Lower

16 Xfr to SynLow

20 Iodine

22 SynUpr to Waste

25 Syn Col B Upper

36 IODINE to Syn C 13 Syn Col C Lower

16 Xfr to SynLow

20 Iodine

22 SynUpr to Waste

24 Syn Col C Upper

37 IODINE to Waste 20 Iodine

21 SynLow to Waste

38 TCA to Syn Cols 13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

16 Xfr to SynLow

19 TCA

23 SynUpr to Trityl

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

39 TCA to SynCol A 15 Syn Col A Lower

16 Xfr to SynLow

19 TCA

23 SynUpr to Trityl

26 Syn Col A Upper




40 TCA to SynCol B 14 Syn Col B Lower

16 Xfr to SynLow

19 TCA

23 SynUpr to Trityl

25 Syn Col B Upper

41 TCA to SynCol C 13 Syn Col C Lower

16 Xfr to SynLow

19 TCA

23 SynUpr to Trityl

24 Syn Col C Upper

42 TCA to Trityl 19 TCA

21 SynLow to Waste

72 SynLow to Trityl

43 ACN to Syn Cols 1 ACN to Syn

13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

22 SynUpr to Waste

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

44 ACN to SynCol A 1 ACN to Syn

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper

45 ACN to SynCol B 1 ACN to Syn

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper

46 ACN to SynCol C 1

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper

47 CN to SynWaste 1 ACN to Syn

16 Xfr to SynLow

21 SynLow to Waste

48 TET to Syn Cols 4 Tetrazole

13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

22 SynUpr to Waste




24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

49 TET to SynCol A 4 Tetrazole

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper

50 TET to SynCol B 4 Tetrazole

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper

51 TET to Syn Col C 4 Tetrazole

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper

52 TET to Waste 4 Tetrazole

16 Xfr to SynLow

21 SynLow to Waste

53 Flush Syn Cols 2 Gas to Syn

13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

22 SynUpr to Waste

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

54 Flush Syn Col A 2 Gas to Syn

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper

55 Flush Syn Col B 2 Gas to Syn

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper

56 Flush Syn Col C 2 Gas to Syn

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper

57 Back Flush Syns 13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower




16 Xfr to SynLow

21 SynLow to Waste

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

28 Gas to Syn Upper

58 Back Flush Syn A 15 Syn Col A Lower

16 Xfr to SynLow

21 SynLow to Waste

26 Syn Col A Upper

28 Gas to Syn Upper

59 Back Flush Syn B 14 Syn Col B Lower

16 Xfr to SynLow

21 SynLow to Waste

25 Syn Col B Upper

28 Gas to Syn Upper

60 Back Flush Syn C 13 Syn Col C Lower

16 Xfr to SynLow

21 SynLow to Waste

24 Syn Col C Upper

28 Gas to Syn Upper

61 TritylFlsh Syns 2 Gas to Syn

13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

23 SynUpr to Trityl

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

62 TritylFlsh SynA 2 Gas to Syn

15 Syn Col A Lower

23 SynUpr to Trityl

26 Syn Col A Upper

63 TritylFlsh SynB 2 Gas to Syn

14 Syn Col B Lower

23 SynUpr to Trityl

25 Syn Col B Upper

64 TritylFlsh SynC 2 Gas to Syn

13 Syn Col C Lower

23 SynUpr to Trityl

24 Syn Col C Upper




65 SynLowBlk Flush 2 Gas to Syn

16 Xfr to SynLow

21 SynLow to Waste

66 Syn Upper Vent 22 SynUpr to Waste

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

67 Syn Lower Vent 13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

16 Xfr to SynLow

21 SynLow to Waste

68 ACN to AGCT 1 ACN to Syn

9 “A” Amidite

10 “G” Amidite

11 “C” Amidite

12 “T” Amidite

76 Amidite Vent

69 ACN to TET 1 ACN to Syn

4 Tetrazole

70 ACN to TCA 1 ACN to Syn

16 Xfr to SynLow

19 TCA

71 ACN to IODINE 1 ACN to Syn

16 Xfr to SynLow

20 Iodine

72 ACN to AC20 1 ACN to Syn

16 Xfr to SynLow

18 Ac20

73 ACN to NMI 1 ACN to Syn

16 Xfr to SynLow

17 NMI

74 A AMIDITE Waste 9 "A" Amidite

16 Xfr to SynLow

21 SynLow to Waste

75 G AMIDITE Waste 10 "G" Amidite

16 Xfr to SynLow

21 SynLow to Waste




D 3948 Function List and Other Cycles



76 C AMIDITE Waste 11 "C" Amidite

16 Xfr to SynLow

21 SynLow to Waste

77 T AMIDITE Waste 12 "T" Amidite

16 Xfr to SynLow

21 SynLow to Waste

78 5 AMIDITE Waste 5 "5" Amidite

16 Xfr to SynLow

21 SynLow to Waste


16 Xfr to SynLow

21 SynLow to Waste


16 Xfr to SynLow

21 SynLow to Waste


16 Xfr to SynLow

21 SynLow to Waste

82 ACN AMIDITE A 1 ACN to Syn

9 "A" Amidite

76 Amidite Vent

83 ACN AMIDITE G 1 ACN to Syn

10 "G" Amidite

76 Amidite Vent

84 ACN AMIDITE C 1 ACN to Syn

11 "C" Amidite

76 Amidite Vent

85 ACN AMIDITE T 1 ACN to Syn

12 "T" Amidite

76 Amidite Vent

86 ACN AMIDITE 5 1 ACN to Syn

5 "5" Amidite

76 Amidite Vent


6 "6" Amidite

76 Amidite Vent


7 "7" Amidite

76 Amidite Vent



8 "8" Amidite

76 Amidite Vent

90 Mix AMIDITE A 2 Gas to Syn

9 "A" Amidite

76 Amidite Vent

91 Mix AMIDITE G 2 Gas to Syn

10 "G" Amidite

76 Amidite Vent

92 Mix AMIDITE C 2 Gas to Syn

11 "C" Amidite

76 Amidite Vent

93 Mix AMIDITE T 2 Gas to Syn

12 "T" Amidite

76 Amidite Vent

94 Mix AMIDITE 5 2 Gas to Syn

5 "5" Amidite

76 Amidite Vent


6 "6" Amidite

76 Amidite Vent


7 "7" Amidite

76 Amidite Vent


8 8" Amidite

76 Amidite Vent

98 Mix AGCT 2 Gas to Syn

9 'A" Amidite

10 "G" Amidite

11 "C" Amidite

12 "T" Amidite

76 Amidite Vent

99 MIX AG 2 Gas to Syn

9 "A" Amidite

10 "G" Amidite

76 Amidite Vent

100 MIX CT 2 Gas to Syn

11 "C" Amidite

12 "T" Amidite

76 Amidite Vent

101 AMIDITE Vent 76 Amidite Vent




102 Ammonia Vent 74 Ammonia Vent

103 Mix Ammonia 29 Gas to Clv Lower

32 Ammonia

74 Ammonia Vent

104 Mix Tetrazole 2 Gas to Syn

4 Tetrazole

105 SynUprBlk Flush 22 SynUpr to Waste

28 Gas to Syn Upper

106 LowTrityl Flush 2 Gas to Syn

16 Xfr to SynLow

21 SynLow to Waste

72 SynLow to Trityl

107 UprTrityl Flush 23 SynUpr to Trityl

28 Gas to Syn Upper

108 ACN to Trityl 1 ACN to Syn

16 Xfer to SynLow

21 SynLow to Waste

72 SynLow to Trityl

109 Waste to Trityl

Numb Name Purpose of Function

110 Database Version This function is used to indicate the database version.

111 Ammonia to Clvs 32 Ammonia

33 Clv Col C Lower

34 Clv Col B Lower

35 Clv Col A Lower

112 Ammonia to ClvA 32 Ammonia

35 Clv Col A Lower

113 Ammonia to ClvB 32 Ammonia

34 Clv Col B Lower

114 Ammonia to ClvC 32 Ammonia

33 Clv Col C Lower

115 Ammonia toWaste 32 Ammonia

39 Xfr Clv to Dep

43 DepLow to Waste

116 ACN to Clv Cols 33 Clv Col C Lower

34 Clv Col B Lower

35 Clv Col A Lower

38 ACN to Dep Lower

39 Xfr Clv to Dep

117 ACN to ClvCol A 35 Clv Col A Lower




38 ACN to Dep Lower

39 Xfr Clv to Dep

118 ACN to ClvCol B 34 Clv Col B Lower

38 ACN to Dep Lower

39 Xfr Clv to Dep

119 ACN to ClvCol C 33 Clv Col C Lower

38 ACN to Dep Lower

39 Xfr Clv to Dep

120 ACN to ClvWaste 38 ACN to Dep Lower

43 DepLow to Waste

121 Gas to Clv Cols 29 Gas to Clv Lower

33 Clv Col C Lower

34 Clv Col B Lower

35 Clv Col A Lower

122 Gas to ClvCol A 29 Gas to Clv Lower

35 Clv Col A Lower

123 Gas to ClvCol B 29 Gas to Clv Lower

34 Clv Col B Lower

124 Gas to ClvCol C 29 Gas to Clv Lower

33 Clv Col C Lower

125 Gas to Clv II 33 Clv Col C Lower

34 Clv Col B Lower

35 Clv Col A Lower

37 Gas to Dep Lower

39 Xfr Clv to Dep

126 H2O to Clv Cols 31 H2O to Clv Lower

33 Clv Col C Lower

34 Clv Col B Lower

35 Clv Col A Lower

127 H2O to ClvCol A 31 H2O to Clv Lower

35 Clv Col A Lower

128 H2O to ClvCol B 31 H2O to Clv Lower

34 Clv Col B Lower

129 H2O to ClvCol C 31 H2O to Clv Lower

33 Clv Col C Lower

130 H2O to ClvWaste 31 H2O to Clv Lower

39 Xfr Clv to Dep

43 DepLow to Waste

131 Back Flush Clvs 33 Clv Col C Lower

34 Clv Col B Lower




35 Clv Col A Lower

39 Xfr Clv to Dep

43 DepLow to Waste

75 Gas to Clv Upper

132 Back flush Clv A 35 Clv Col A Lower

39 Xfr Clv to Dep

43 DepLow to Waste

75 Gas to Clv Upper

133 Back flush Clv B 34 Clv Col B Lower

39 Xfr Clv to Dep

43 DepLow to Waste

75 Gas to Clv Upper

134 Back flush Clv C 33 Clv Col C Lower

39 Xfr Clv to Dep

43 DepLow to Waste

75 Gas to Clv Upper

135 Crude A to UV 37 Gas to Dep Lower

40 Coil A Input

46 Coil A Exit

49 Xfr from Dep

58 Xfr from Dep

62 Xfr to UV

136 Crude B to UV 37 Gas to Dep Lower

41 Coil B Input

47 Coil B Exit

49 Xfr from Dep

58 Xfr from Dep

62 Xfr to UV

137 Crude C to UV 37 Gas to Dep Lower

42 Coil C Input

48 Coil C Exit

49 Xfr from Dep

58 Xfr from Dep

62 Xfr to UV

138 Press Line 29 Gas to Clv Lower

139 Compress 75 Gas to Clv Upper

140 Clv Vent 33 Clv Col C Lower

34 Clv Col B Lower

35 Clv Col A Lower

39 Xfr Clv to Dep

43 DepLow to Waste




141 Xfer Clv Col A 35 Clv Col A Lower

39 Xfr Clv to Dep

40 Coil A Input

46 Coil A Exit

50 DepUpr to Waste

75 Gas to Clv Upper

Note Functions 141, 142, and 143 “ramp up” the pressure from the regulator until the sample leaves the coil during a cleavage cycle. In examining the cycle in the Edit Cleavage Cycle view, the value in the MISC field is the regulator being ramped and theTIME field is the time between ramps. These functions will ramp up to four times before quitting. Ramping only works when “Yes” is selected for the SNS field.

To observe the range of pressure over which one of these functions is ramped for a particular regulator, change to the Monitor Instrument view while the function is being executed.

The following functions are also ramping functions:

� 135–137 Crude to UV

� 162-168 Xfer Coil

� 211-213 Pur to UV

142 Xfer Clv Col B 34 Clv Col B Lower

39 Xfr Clv to Dep

41 Coil B Input

47 Coil B Exit

50 DepUpr to Waste

75 Gas to Clv Upper

143 Xfer Clv Col C 33 Clv Col C Lower

39 Xfr Clv to Dep

42 Coil C Input

48 Coil C Exit

50 DepUpr to Waste

75 Gas to Clv Upper

144 Clv Blk Flush 29 Gas to Clv Lower

39 Xfr Clv to Dep

43 DepLow to Waste







145 Ammonia to SynA 3 Xfer from Syn

15 Syn Col A Lower

22 SynUpr to Waste

26 Syn Col A Upper

30 Xfr to Clv Lower

32 Ammonia

146 Ammonia to SynB 3 Xfer from Syn

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper

30 Xfr to Clv Lower

32 Ammonia

147 Ammonia to SynC 3 Xfer from Syn

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper

30 Xfr to Clv Lower

32 Ammonia

148 Syn ACN to Clv 1 ACN to Syn

3 Xfer from Syn

30 Xfr to Clv Lower

39 Xfer Clv to Dep

43 DepLow to Waste

149 Flsh Syn to Clv 2 Gas to Syn

3 Xfer from Syn

30 Xfr to Clv Lower

39 Xfer Clv to Dep

43 DepLow to Waste

150 *User Fxn 150 *

151 Dep Xfer Rinse 31 H2O to Clv Lower

39 Xfr Clv to Dep

43 DepLow to Waste

152 Dep Xfer Flush 29 Gas to Clv Lower

39 Xfr Clv to Dep

43 DepLow to Waste

153 DepLowBlk Flush 37 Gas to Dep Lower

43 DepLow to Waste

154 DepUprBlk Rinse 45 H2O to Dep Upper

50 DepUpr to Waste

155 Gas to Dep Coils 37 Gas to Dep Lower


40 Coil A Input

41 Coil B Input

42 Coil C Input

46 Coil A Exit

47 Coil B Exit

48 Coil C Exit

50 DepUpr to Waste

156 Gas to Coil A 37 Gas to Dep Lower

40 Coil A Input

46 Coil A Exit

50 DepUpr to Waste

157 Gas to Coil B 37 Gas to Dep Lower

41 Coil B Input

47 Coil B Exit

50 DepUpr to Waste

158 Gas to Coil C 37 Gas to Dep Lower

42 Coil C Input

48 Coil C Exit

50 DepUpr to Waste

159 ACN to Coil A 38 ACN to Dep Lower

40 Coil A Input

46 Coil A Exit

50 DepUpr to Waste

160 ACN to Coil B 38 ACN to Dep Lower

41 Coil B Input

47 Coil B Exit

50 DepUpr to Waste

161 ACN to Coil C 38 ACN to Dep Lower

42 Coil C Input

48 Coil C Exit

50 DepUpr to Waste

162 H2O toDep Coils 40 Coil A Input

41 Coil B Input

42 Coil C Input

43 DepLow to Waste

45 H2O to Dep Upper

46 Coil A Exit

47 Coil B Exit

48 Coil C Exit

163 H2O to Coil A 40 Coil A Input

43 DepLow to Waste




45 H2O to Dep Upper

46 Coil A Exit

164 H2O to Coil B 41 Coil B Input

43 DepLow to Waste

45 H2O to Dep Upper

47 Coil B Exit

165 H2O to Coil C 42 Coil C Input

43 DepLow to Waste

45 H2O to Dep Upper

48 Coil C Exit

Note Functions 166, 167, and 168 “ramp up” the pressure from the regulator until the sample is transferred during a purification cycle. In examining the cycle in the Edit Purification Cycle view, the value in the MISC field is the regulator being ramped.

To observe the range of pressure over which one of these functions is ramped for a particular regulator, change to the Monitor Instrument view while the function is being executed.

See material on page B-5 for more information.

166 Xfr Coil A 37 Gas to Dep Lower

40 Coil A Input

46 Coil A Exit

49 Xfr from Dep

58 Xfr from Dep

61 Pur Col A Lower

67 Pur Col A Upper

68 PurUpr to Waste

167 Xfr Coil B 37 Gas to Dep Lower

41 Coil B Input

47 Coil B Exit

49 Xfr from Dep

58 Xfr from Dep

60 Pur Col B Lower

66 Pur Col B Upper

68 PurUpr to Waster

168 Xfr Coil C 37 Gas to Dep Lower

42 Coil C Input

48 Coil C Exit

49 Xfr from Dep

58 Xfr from Dep

59 Pur Col C Lower

65 Pur Col C Upper




68 PurUpr to Waster

169 Depro Htr Wait MISC field states how long the heater must be hot (in whole minutes). If the heater hasn’t been hot at least that long, the time the function must wait (in seconds) is calculated and the heater stays hot for the calculated time.

170 Start Depro Htr Starts the heater ramping up to the temperature specified in the MISC field. Deprotection time begins once the heater reaches the specified temperature.

171 Pur Xfer Rinse 45 H2O to Dep Upper

49 Xfr from Dep

58 Xfr from Dep

63 PurLow to Waste

172 Pur Xfer Flush 44 Gas to Dep Upper

49 Xfr from Dep

58 Xfr from Dep

63 PurLow to Waste

173 ACN to Pur Cols 52 ACN to Pur

59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

65 Pur Col C Upper

66 Pur Col B Upper

67 Pur Col A Upper

68 PurUpr to Waste

174 ACN Pur Col A 52 ACN to Pur

61 Pur Col A Lower

67 Pur Col A Upper

68 PurUpr to Waste

175 ACN Pur Col B 52 ACN to Pur

60 Pur Col B Lower

66 Pur Col B Upper

68 PurUpr to Waste

176 ACN Pur Col C 52 ACN to Pur

59 Pur Col C Lower

65 Pur Col C Upper

68 PurUpr to Waste


177 ACN to PurWaste 52 ACN to Pur

63 Pur Low to Waste




178 TEAA to Pur Cols 53 TEAA

59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

65 Pur Col C Upper

66 Pur Col B Upper

67 Pur Col A Upper

68 PurUpr to Waste

179 TEAA to Pur A 53 TEAA

61 Pur Col A Lower

67 Pur Col A Upper

68 PurUpr to Waste

180 TEAA to Pur B 53 TEAA

60 Pur Col B Lower

66 Pur Col B Upper

68 PurUpr to Waste

181 TEAA to Pur C 53 TEAA

59 Pur Col C Lower

65 Pur Col C Upper

68 PurUpr to Waste

182 TEAA to Waste 53 TEAA

63 Pur Low to Waste

183 H2O to Pur Cols 56 H2O to Pur

59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

65 Pur Col C Upper

66 Pur Col B Upper

67 Pur Col A Upper

68 PurUpr to Waste

184 H2O to Pur Col A 56 H2O to Pur

61 Pur Col A Lower

67 Pur Col A Upper

68 PurUpr to Waste

185 H2O to Pur Col B 56 H2O to Pur

60 Pur Col B Lower

66 Pur Col B Upper


68 PurUpr to Waste

186 H2O to Pur Col C 56 H2O to Pur




59 Pur Col C Lower

65 Pur Col C Upper

68 PurUpr to Waste

187 H2O to PurWaste 56 H2O to Pur

63 Pur Low to Waste

188 TFA to Pur Cols 54 TFA

59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

65 Pur Col C Upper

66 Pur Col B Upper

67 Pur Col A Upper

68 PurUpr to Waste

189 TFA to PurCol A 54 TFA

61 Pur Col A Lower

67 Pur Col A Upper

68 PurUpr to Waste

190 TFA to PurCol B 54 TFA

60 Pur Col B Lower

66 Pur Col B Upper

68 PurUpr to Waste

191 TFA to PurCol C 54 TFA

59 Pur Col C Lower

65 Pur Col C Upper

68 PurUpr to Waste

192 TFA to Waste 54 TFA

63 Pur Low to Waste

193 20%ACN to Purs 57 20% ACN

59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

65 Pur Col C Upper

66 Pur Col B Upper

67 Pur Col A Upper

68 PurUpr to Waste

194 20%ACN to Pur A 57 20% ACN

61 Pur Col A Lower

67 Pur Col A Upper

68 PurUpr to Waste





195 20%ACN to Pur B 57 20% ACN

60 Pur Col B Lower

66 Pur Col B Upper

68 PurUpr to Waste

196 20%ACN to Pur C 57 20% ACN

59 Pur Col C Lower

65 Pur Col C Upper

68 PurUpr to Waste

197 20% ACN to Waste 57 20% ACN

63 Pur Low to Waste

198 AcAcid to Purs 55 Acetic Acid

59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

65 Pur Col C Upper

66 Pur Col B Upper

67 Pur Col A Upper

68 PurUpr to Waste

199 AcAcid to Pur A 55 Acetic Acid

61 Pur Col A Lower

67 Pur Col A Upper

68 PurUpr to Waste

200 AcAcid to Pur B 55 Acetic Acid

60 Pur Col B Lower

66 Pur Col B Upper

68 PurUpr to Waste

201 AcAcid to Pur C 55 Acetic Acid

59 Pur Col C Lower

65 Pur Col C Upper

68 PurUpr to Waste

202 AcAcid to Waste 55 Acetic Acid

63 PurLow to Waste

203 Gas to Pur Cols 51 Gas to Pur

59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

65 Pur Col C Upper

66 Pur Col B Upper

67 Pur Col A Upper

68 PurUpr to Waste

204 Gas to PurCol A 51 Gas to Pur




61 Pur Col A Lower

67 Pur Col A Upper

68 PurUpr to Waste

205 Gas to PurCol B 51 Gas to Pur

60 Pur Col B Lower

66 Pur Col B Upper

68 PurUpr to Waste

206 Gas to PurCol C 51 Gas to Pur

59 Pur Col C Lower

65 Pur Col C Upper

68 PurUpr to Waste

207 Back Flush Purs 59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

63 Pur Low to Waste

65 Pur Col C Upper

66 Pur Col B Upper

67 Pur Col A Upper

69 Gas to Pur Upper

208 Back Flsh Pur A 61 Pur Col A Lower

63 Pur Low to Waste

67 Pur Col A Upper

69 Gas to Pur Upper

209 Back Flsh Pur B 60 Pur Col B Lower

63 Pur Low to Waste

66 Pur Col B Upper

69 Gas to Pur Upper

210 Back Flsh Pur C 59 Pur Col C Lower

63 Pur Low to Waste

65 Pur Col C Upper

69 Gas to Pur Upper

211 Pur A to UV 61 Pur Col A Lower

62 Xfr to UV

67 Pur Col A Upper

69 Gas to Pur Upper

212 Pur B to UV 60 Pur Col B Lower

62 Xfr to UV

66 Pur Col B Upper

69 Gas to Pur Upper







213 Pur C to UV 59 Pur Col C Lower

62 Xfr to UV

65 Pur Col C Upper

69 Gas to Pur Upper

214 H2O to UV 56 H2O to Pur

62 Xfr to UV

215 Xfer UV to SC 77 UV Lower

78 Gas to UV

216 UV Reading

217 Flush UV toWaste 77 UV Lower

78 Gas to UV

79 UV to Waste

218 PurLowBlk Flush 51 Gas to Pur

63 Pur Low to Waste

219 PurUprBlk Flush 68 PurUpr to Waste

69 Gas to Pur Upper

220 Pur Vent 59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

63 Pur Low to Waste

221 DepUprBlk Flush 44 Gas to Dep Upper

50 DepUpr to Waste

222 SC Block Flush 51 Gas to Pur

62 Xfr to UV

223 Set UV Scale Adjusts ODU calculation with a scaling factor expressed as a percent.

224 Pur Gas to Dep 49 Xfer from Dep

50 DepUpr to Waste

51 Gas to Pur

58 Xfer from Dep

225 Pur ACN to Dep 49 Xfer from Dep

50 DepUpr to Waste

52 ACN to Pur

58 Xfer from Dep

226 Pur H2O to Dep 49 Xfer from Dep

50 DepUpr to Waste

56 H2O to Pur

58 Xfer from Dep

227 *User Fxn 227 *

228 *User Fxn 228 *


229 End Row SCP/123

230 Go Sub On Fail

231 Syn Jaw Open

232 Syn Jaw Close 2 Gas to Syn

13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

16 Xfer to SynLow

28 Gas to Syn Upper

233 Clv Jaw Open

234 Clv Jaw Close 29 Gas to Clv Lower

39 Xfer Clv to Dep

75 Gas to Clv Upper

235 Pur Jaw Open

236 Pur Jaw Close 49 Xfer from Dep

51 Gas to Pur

58 Xfer from Dep

59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

69 Gas to Pur Upper

237 Open All Jaws

238 Close All Jaws

239 TurnTable Home

240 TurnTable Next

241 TurnTable Prev

242 SC Waste Puts needle over sample vials.

243 SC Home Sends tray to “home” position.

244 SC Next Sends tray to the next position.

245 SC Open Sends tray to the front.

246 SC Close Sends tray to correct position according to index.

247 SC Needle Up

248 SC Needle Down

249 Relay 1 On

250 Relay 1 Off

251 Relay 1 Pulse

252 Relay 2 On

253 Relay 2 Off

254 Relay 2 Pulse

255 Fan On

256 Fan Off



257 Heater On

258 Heater Off

259 Wait

260 Set Coil Temp

261 Wait Coil Temp Cycle waits for heater to reach temp. in the Misc field, cycle resumes when temp. is reached or when step times out. On timeout, temp. reached before going on is reported in the status window (Monitor Chemistry).

262 Tggle Vlves On Turns single valve ON until Tggle Vlves Off function is used.

263 Tggle Vlves Off Used to turn OFF single valves activated by the Tggle Vlves On function.

264 SC Prev Sends tray to the previous position according to index.

265 Set Rmp Fxn Sns These functions are used to set parameters which help define the behavior of the ramping functions. See 265 Set Rmp Fxn Sns, et al in Appendix A.

266 Set Rmp Fxn Trp

267 Set Rmp Fxn Chk

268 Set Rmp Fxn Dly

269 Send Message These two parameters are used to send a message as specified in the MISC field. See 269 Send Message/ 270Comment in Appendix A.

270 Comment

271 Begin of Cycle These functions serve only to mark the beginning and ending steps of a cycle procedure.

272 End of Cycle

273 Begin Loop These functions mark the beginning and ending boundaries of a block of steps to be executed repeatedly.

274 End Loop

275 Start Here “cycle entry” - First Synthesis cycle skips to this step to begin.

276 Exit DMT On This step executes only on the last synthesis cycle of any given column. See the discussion in Appendix A.

277 If Pur Col A Execute steps to Else or Endif if Col A is active, otherwise skips steps.

278 If Pur Col B Execute steps to Else or Endif if Col B is active, otherwise skips steps.

279 If Pur Col C Execute steps to Else or Endif if Col C is active, otherwise skips steps.

280 Else Execute when previous If statement condition is false;

281 Endif End of If statement.

282 Select Pur Cols Picks columns that require purification (no Crude).

283 Select Act Cols Picks active columns, crude or pure.



284 Clv Wants Depro Cleavage module waits on Pur flag reset.

285 Pure Owns Depro (I) - sets flag at the beginning to “Pur.” Flag indicates that purification “owns” deprotection coils.

286 Clv Owns Depro (O) - resets flag at the end to “Not Pur ” - releases deprotection coils to cleavage.

287 Go Sub

288 Return Sub

289 Sub Label

290 Goto End

291 If Any Act Cols Executes steps to nearest Else or Endif, only if there are columns purifying - deactivates all non-purifying columns.

292 Go Sub A Do subroutine (Go Sub A) only if column is active. Deactivates other columns until Return Sub.

293 Go Sub B Do subroutine (Go Sub B) only if column is active. Deactivates other columns until Return Sub.

294 Go Sub C Do subroutine (Go Sub C) only if column is active. Deactivates other columns until Return Sub.

295 If First Cycle

296 If Last Cycle

297 If Crude Col A

298 If Crude Col B

299 If Crude Col C

300 Engage All Cols

301 Set Pres Reg 1

302 Set Pres Reg 2

303 Set Pres Reg 3

304 Set Pres Reg 4

305 Set Pres Reg 5

306 Set Pres Reg 6

307 Set Pres Reg 7

308 Set Pres Reg 8

309 Set Pres Reg 9

310 Set Pres Reg 10

311 Set Press RegAll

312 Press Reg On

313 Press Reg Off

314 Pres Reg AllOn






315 Pres Reg AllOff

316 If Cyc Greater

317 Save Reg Pres

318 Restore RegPres

319 Revive Act Cols

320 Check Snsr Cal Checks all sensor calibrations. “Wet readings” are at least 2X “dry readings.”

321 CalibrateGas Sns Takes a dry sensor calibration reading.

322 CalibrateLiqSns Takes a wet sensor calibration reading.

323 Calib AllGasSns

324 Calib AllLiqSns

325 Jaw Test Time Used during instrument assembly.

326 Pres SynJaws 24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

28 Gas to Syn Upper

327 Pres ClvJaws 75 Gas to Clv Upper

328 Pres PurJaws 51 Gas to Pur

59 Pur Col C Lower

60 Pur Col B Lower

61 Pur Col A Lower

329 Begin Leak Test

330 End Leak Test

331 NMI to Cols 13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

16 Xfr to SynLow

17 NMI

22 SynUpr to Waste

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

332 NMI to Col A 15 Syn Col A Lower

16 Xfr to SynLow

17 NMI

22 SynUpr to Waste

26 Syn Col A Upper

333 NMI to Col B 14 Syn Col B Lower

16 Xfr to SynLow

17 NMI

22 SynUpr to Waste


25 Syn Col B Upper

334 NMI to Col C 13 Syn Col C Lower

16 Xfr to SynLow

17 NMI

22 SynUpr to Waste

24 Syn Col C Upper

335 AC20 to Cols 13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

16 Xfr to SynLow

18 Ac20

22 SynUpr to Waste

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

336 AC20 to Col A 15 Syn Col A Lower

16 Xfr to SynLow

18 Ac20

22 SynUpr to Waste

26 Syn Col A Upper

337 AC20 to Col B 14 Syn Col B Lower

16 Xfr to SynLow

18 Ac20

22 SynUpr to Waste

25 Syn Col B Upper

338 AC20 to Col C 13 Syn Col C Lower

16 Xfr to SynLow

18 Ac20

22 SynUpr to Waste

24 Syn Col C Upper

339 Tet to Test 4 Tetrazole

13 Syn Col C Lower

14 Syn Col B Lower

15 Syn Col A Lower

22 SynUpr to Waste

24 Syn Col C Upper

25 Syn Col B Upper

26 Syn Col A Upper

340 Tet to Test A 4 Tetrazole

15 Syn Col A Lower

22 SynUpr to Waste




26 Syn Col A Upper

341 Tet to Test B 4 Tetrazole

14 Syn Col B Lower

22 SynUpr to Waste

25 Syn Col B Upper

342 Tet to Test C 4 Tetrazole

13 Syn Col C Lower

22 SynUpr to Waste

24 Syn Col C Upper

343 A to Col B 9 "A" Amidite

14 Syn Col B Lower

22 Syn Upr to Waste

25 Syn Col B Upper

344 G to Col B 10 "G" Amidite

14 Syn Col B Lower

22 Syn Upr to Waste

25 Syn Col B Upper

345 C to Col B 11 "C" Amidite

14 Syn Col B Lower

22 Syn Upr to Waste

25 Syn Col B Upper

346 T to Col B 12 "T" Amidite

14 Syn Col B Lower

22 Syn Upr to Waste

25 Syn Col B Upper

347 5 to Col B 5 "5" Amidite

14 Syn Col B Lower

22 Syn Upr to Waste

25 Syn Col B Upper


14 Syn Col B Lower

22 Syn Upr to Waste

25 Syn Col B Upper


14 Syn Col B Lower

22 Syn Upr to Waste

25 Syn Col B Upper


14 Syn Col B Lower

22 Syn Upr to Waste

25 Syn Col B Upper




351 Fish SynA to SC 3 Xfr from Syn

15 Syn Col A Lower

26 Syn Col A Upper

28 Gas to Syn Upper

30 Xfr to Clv Lower

36 Mfg Test 36

55 Acetic Acid

62 Xfr to UV

352 Flsh SynB to SC 3 Xfr from Syn

14 Syn Col B Lower

25 Syn Col B Upper

28 Gas to Syn Upper

30 Xfr to Clv Lower

36 Mfg Test 36

55 Acetic Acid

62 Xfr to UV

353 Flsh SynC to SC 3 Xfr from Syn

13 Syn Col C Lower

24 Syn Col C Upper

28 Gas to Syn Upper

30 Xfr to Clv Lower

36 Mfg Test 36

55 Acetic Acid

62 Xfr to UV

354 Flsh ClvA to SC 35 Clv Col A Lower

55 Acetic Acid

62 Xfr to UV

75 Gas to Clv Upper

36 Mfg Test 36

355 Flsh ClvB to SC 34 Clv Col B Lower

55 Acetic Acid

62 Xfr to UV

75 Gas to Clv Upper

36 Mfg Test 36

356 Flsh ClvC to SC 33 Clv Col C Lower

55 Acetic Acid

62 Xfr to UVr

75 Gas to Clv Uppe

36 Mfg Test v36

357 ACN to Ammonia 32 Ammonia

38 ACN to Dep Lower




39 Xfr Clv to Dep

358 ACN to TEAA 52 ACN to Pur

53 TEAA

359 ACN to TFA 52 ACN to Pur

54 TFA

360 ACN to H2O(1) 52 ACN to Pur

56 H2O to Pur

361 ACN to H2O(2) 31 H2O to Clv Lower

38 ACN to Dep Lower

39 Xfr Clv to Dep

362 ACN to H2O(3) 38 ACN to Dep Lower

40 Coil A Input

45 H2O to Dep Upper

46 Coil A Exit

363 ACN to 20%ACN 52 ACN to Pur

57 20% ACN

364 ACN to V23 1 ACN to Syn

13 Syn Col C Lower

23 SynUpr to Trityl

24 Syn Col C Upper

365 ACN to V70 NC 52 ACN to Pur

63 Pur Low to Waste

70 Xfr to Trityl

366 ACN to SC NC 52 ACN to Pur

62 Xfr to UVr

367 ACN to V43 1 ACN to Syn

3 Xfr from Syn

30 Xfr to Clv Lower

39 Xfr Clv to Dep

43 Dep Low to Waste

368 ACN to V70 NO 52 ACN to Pur

63 Pur Low to Waste

369 ACN to V79 NC 52 ACN to Pur

62 Xfr to UV

77 UV Lower

79 UV to Waste

370 ACN to DEPRO A 38 ACN to Dep Lower

40 Coil A Input

46 Coil A Exit

50 Dep Upr to Waste




371 ACN to DEPRO B 38 ACN to Dep Lower

41 Coil B Input

47 Coil B Exit

50 Dep Upr to Waste

372 ACN to DEPRO C 38 ACN to Dep Lower

42 Coil C Input

48 Coil C Exit

50 Dep Upr to Waste

373 R3-UV-SC 51 Gas to Pur

62 Xfr to UV

77 UV Lower

78 Gas to UV

374 R3-UV-Waste 51 Gas to Pur

62 Xfr to UV

77 UV Lower

78 Gas to UV

79 UV to Waste

375 ACN to AcAcid 55 Acetic Acid

52 ACN to Pur

376 Depro Xfer SC 36 Mfg Test v36

37 Gas to De Lower

39 Xfr Clv to Dep

55 Acetic Acid

62 Xfr to UV

377 Flsh DepA to SC 37 Gas to Dep Lower

40 Coil A Input

46 Coil A Exit

49 Xfr from Dep

58 Xfr from Dep

62 Xfr to UV

378 Flsh DepB to SC 37 Gas to Dep Lower

41 Coil B Input

47 Coil B Exit

49 Xfr from Dep

58 Xfr from Dep

62 Xfr to UV

379 Flsh DepC to SC 37 Gas to Dep Lower

42 Coil C lnput

48 Coil C Exit

49 Xfr from Dep

58 Xfr from Dep




62 Xfr to UV

380 *User Fxn 380 *

381 *User Fxn 381 *

382 *User Fxn 382 *

383 *User Fxn 383 *

384 *User Fxn 384 *

385 *User Fxn 385 *

386 *User Fxn 386 *

387 *User Fxn 387 *

388 *User Fxn 388 *

389 *User Fxn 389 *

390 *User Fxn 390 *

391 *User Fxn 391 *

392 *User Fxn 392 *

393 *User Fxn 393 *

394 *User Fxn 394 *

395 Pause Chemistry

396 Time Stamp Report current instrument time with timestamp # to Microphone.

397 Test Valves

398 Depro Test

399 If Test To Do Execute cycle steps to Endif if Test is selected (the test to perform is coded in the Misc field.

400 * Reserved *

401 SynUpr Wet 401

402 SynUprWet A 402

403 SynUprWet B 403

404 SynUprWet C 404

405 SynUpr Wet 405

406 SynUprWet A 406

407 SynUprWet B 407

408 SynUprWet C 408

409 SynUpr Wet 409

410 SynUprWet A 410

411 SynUprWet B 411

412 SynUprWet C 412

413 SynUpr Dry 413

414 SynUprDry A 414

415 SynUprDry B 415

416 SynUprDry C 416




417 SynMan Wet 417

418 SynManWet A 418

419 SynManWet B 419

420 SynManWet C 420

421 SynMan Wet 421

422 SynManWet A 422

423 SynManWet B 423

424 SynManWet C 424

425 SynMan Wet 425

426 SynManWet A 426

427 SynManWet B 427

428 SynManWet C 428

429 SynLow Wet 429

430 SynLowWet A 430

431 SynLowWet B 431

432 SynLowWet C 432

433 SynLow Dry 433

434 SynLowDry A 434

435 SynLowDry B 435

436 SynLowDry C 436

437 Clv Wet 437

438 Clv Wet A 438

439 Clv Wet B 439

440 Clv Wet C 440

441 Clv Wet 441

442 Clv Wet A 442

443 Clv Wet B 443

444 Clv Wet C 444

445 Clv Dry 445

446 Clv Dry A 446

447 Clv Dry B 447

448 Clv Dry C 448

449 Clv Dry 449

450 Clv Dry A 450

451 Clv Dry B 451

452 Clv Dry C 452

453 Coil Wet 453

454 Coil Wet A 454

455 Coil Wet B 455







456 Coil Wet C 456

457 Coil Dry 457

458 Coil Dry A 458

459 Coil Dry B 459

460 Coil Dry C 460

461 Pur Wet 461

462 Pur Wet A 462

463 Pur Wet B 463

464 Pur Wet C 464

465 Pur Wet 465

466 Pur Wet A 466

467 Pur Wet B 467

468 Pur Wet C 468

469 Pur Wet 469

470 Pur Wet A 470

471 Pur Wet B 471

472 Pur Wet C 472

473 Pur Wet 473

474 Pur Wet A 474

475 Pur Wet B 475

476 Pur Wet C 476

477 Pur Wet 477

478 Pur Wet A 478

479 Pur Wet B 479

480 Pur Wet C 480

481 Pur Dry 481

482 Pur Dry A 482

483 Pur Dry B 483

484 Pur Dry C 484

485 Pur Dry 485

486 Pur Dry A 486

487 Pur Dry B 487

488 Pur Dry C 488

489 UV Wet 489

490 UV Wet 490

491 UV Wet 491

492 UV Wet 492

493 UV Wet 493

494 UV Wet 494

495 UV Wet 495


496 UV Wet 496

497 UV Wet 497

498 UV Dry 498

499 UV Dry 499

500 UV Dry 500




The Purification Dye CycleA

Listing of Dye Cycle D 3948 Function List and Other Cycles

Table D-2 Pur v.4.40 Dye Cycle

STEP FUNCTION NAME NUM TIME MISC SNS SAFE


2 Pur Owns Depro 285 0.0 0 No Yes

3 If Any Act Cols 291 0.0 0 No Yes

4 Else 280 0.0 0 No Yes

5 Wait 259 60.0 0 No Yes

6 Wait 259 60.0 0 No Yes

7 Wait 259 60.0 0 No Yes

8 SC Next 244 0.0 0 No Yes



11 SC Open 245 0.0 0 No Yes

12 Wait Coil Temp 261 0.0 0 No Yes

13 Set Coil Temp 260 0.0 0 No Yes

14 Clv Owns Depro 286 0.0 0 No Yes

15 Goto End 290 0.0 0 No Yes

16 Endif 281 0.0 0 No Yes

17 Pur Jaw Close 236 0.0 0 No Yes


19 Else 280 0.0 0 No Yes

20 Revive Act Cols 319 0.0 0 No Yes

21 Go Sub 287 0.0 11 No Yes

22 Depro Htr Wait 169 0.0 0 No Yes



25 Go Sub 287 0.0 2 No Yes

26 Go Sub 287 0.0 1 No Yes

27 Go Sub 287 0.0 12 No Yes

28 SC Close 246 0.0 0 No Yes

29 SC Needle Down 248 0.0 0 No Yes

30 Go Sub A 292 0.0 6 No Yes

31 Xfer UV to SC 215 15.0 0 No Yes

32 Go Sub 287 0.0 2 No Yes

33 Go Sub 287 0.0 1 No Yes

34 Go Sub 287 0.0 12 No Yes


36 Go Sub B 293 0.0 6 No Yes



38 Go Sub 287 0.0 2 No Yes

39 Go Sub 287 0.0 1 No Yes

40 Go Sub 287 0.0 12 No Yes


42 Go Sub C 294 0.0 6 No Yes


44 Go Sub 287 0.0 2 No Yes

45 Go Sub 287 0.0 12 No Yes






51 Time Stamp 396 0.0 30 No Yes



54 ACN to PurWaste 177 5.0 0 No Yes


56 Go Sub 287 0.0 3 No Yes

57 Go Sub 287 0.0 8 No Yes

58 Go Sub 287 0.0 3 No Yes

59 Select Pur Cols 282 0.0 0 No Yes


61 Go Sub 287 0.0 5 No Yes

62 Go Sub 287 0.0 3 No Yes

63 Go Sub 287 0.0 5 No Yes

64 Go Sub 287 0.0 3 No Yes

65 Go Sub 287 0.0 4 No Yes

66 Go Sub 287 0.0 3 No Yes

67 Go Sub 287 0.0 4 No Yes

68 Go Sub 287 0.0 3 No Yes

69 Go Sub 287 0.0 9 No Yes

70 Go Sub 287 0.0 9 No Yes

71 Go Sub 287 0.0 5 No Yes

72 Go Sub 287 0.0 3 No Yes

73 Go Sub 287 0.0 5 No Yes

74 Go Sub 287 0.0 3 No Yes

75 Begin Loop 273 0.0 2 No Yes

76 ACN to Pur Cols 173 2.2 0 No Yes

77 ACN to Pur Cols 173 15.0 0 Yes Yes

78 H2O to PurCol A 184 5.0 0 No Yes




79 H2O to PurCol B 185 5.0 0 No Yes

80 H2O to PurCol C 186 5.0 0 No Yes

81 Gas to Pur Cols 203 15.0 0 No Yes

82 Go Sub 287 0.0 3 No Yes

83 End Loop 274 0.0 0 No Yes

84 Go Sub 287 0.0 4 No Yes

85 Go Sub 287 0.0 3 No Yes


87 TEAA to Waste 182 5.0 0 No Yes

88 TEAA to PurCols 178 25.0 0 No Yes

89 TEAA to PurCols 178 45.0 0 Yes Yes


91 Gas to PurCol A 204 10.0 0 No Yes

92 Gas to PurCol B 205 10.0 0 No Yes

93 Gas to PurCol C 206 10.0 0 No Yes


95 Back Flsh Pur A 208 20.0 0 No Yes

96 Back Flsh Pur B 209 20.0 0 No Yes

97 Back Flsh Pur C 210 20.0 0 No Yes






103 Go Sub 287 0.0 10 No Yes

104 Endif 281 0.0 0 No Yes

105 Select Act Cols 283 0.0 0 No Yes

106 Go Sub C 294 0.0 2 No Yes

107 Go Sub 287 0.0 11 No Yes







114 Xfer Coil C 168 12.0 1 Yes No

115 Xfer Coil C 168 20.0 0 No No

116 Go Sub B 293 0.0 2 No Yes

117 Xfer Coil B 167 12.0 1 Yes No

118 Xfer Coil B 167 20.0 0 No No

119 Go Sub A 292 0.0 2 No Yes




120 Xfer Coil A 166 12.0 1 Yes No

121 Xfer Coil A 166 20.0 0 No No



124 Go Sub 287 0.0 10 No Yes








132 AcAcid to Waste 202 2.0 0 No Yes

133 AcAcid to Purs 198 3.0 0 No Yes

134 AcAcid to Purs 198 30.0 0 Yes Yes






140 Go Sub A 292 0.0 7 No Yes

141 Go Sub B 293 0.0 7 No Yes

142 Go Sub C 294 0.0 7 No Yes

143 Go Sub 287 0.0 4 No No

144 Go Sub 287 0.0 3 No Yes

145 Go Sub 287 0.0 4 No Yes

146 Go Sub 287 0.0 3 No Yes

147 Begin Loop 273 0.0 3 No No

148 Go Sub 287 0.0 4 No No

149 Go Sub 287 0.0 3 No Yes

150 End Loop 274 0.0 0 No No

151 20%ACN to Waste 197 4.0 0 No Yes

152 20%ACN to Purs 193 6.0 0 No Yes

153 20%ACN to Purs 193 15.0 0 Yes Yes

154 Go Sub 287 0.0 10 No Yes




158 Endif 281 0.0 0 No Yes






161 Go Sub A 292 0.0 1 No Yes

162 Go Sub A 292 0.0 12 No Yes




166 Pur A to UV 211 10.0 3 Yes Yes

167 Pur A to UV 211 20.0 0 No Yes

168 Go Sub A 292 0.0 14 No Yes

169 Go Sub A 292 0.0 15 No Yes

170 Go Sub B 293 0.0 1 No Yes

171 Go Sub B 293 0.0 12 No Yes

172 SC Next 244 0.0 0 No Yes


174 Pur B to UV 212 10.0 3 Yes Yes

175 Pur B to UV 212 20.0 0 No Yes

176 Go Sub B 293 0.0 14 No Yes

177 Go Sub B 293 0.0 16 No Yes

178 Go Sub C 294 0.0 1 No Yes

179 Go Sub C 294 0.0 12 No Yes

180 SC Next 244 0.0 0 No Yes


182 Pur C to UV 213 10.0 3 Yes Yes

183 Pur C to UV 213 20.0 0 No Yes

184 Go Sub C 294 0.0 14 No Yes

185 Go Sub C 294 0.0 17 No Yes

186 Go Sub 287 0.0 12 No Yes

187 SC Next 244 0.0 0 No Yes

188 SC Open 245 0.0 0 No Yes



191 Gas to Pur Cols 203 45.0 0 Yes Yes


193 Go Sub 287 0.0 3 No Yes

194 Go Sub 287 0.0 8 No Yes

195 Go Sub 287 0.0 3 No Yes



198 Sub Label 289 0.0 1 No Yes


200 SC Waste 242 0.0 0 No Yes

201 H2O to PurWaste 187 5.0 0 No Yes




202 H2O to UV 214 5.0 0 No Yes

203 SC Block Flush 222 5.0 0 No Yes

204 Wait 259 10.0 0 No Yes

205 UV Reading 216 2.0 0 No Yes

206 Flsh UV toWaste 217 6.0 0 No Yes

207 H2O to UV 214 1.0 0 No Yes



210 Return Sub 288 0.0 0 No Yes









219 DepLowBlk Flush 153 2.0 0 No No

220 Return Sub 288 0.0 0 No No



223 Back Flush Purs 207 6.0 0 No Yes

224 Back Flush Purs 207 45.0 0 Yes Yes











235 H2O to Pur Cols 183 3.0 0 No Yes

236 H2O to Pur Cols 183 15.0 0 Yes Yes















248 Crude A to UV 135 12.0 1 Yes Yes

249 Crude A to UV 135 20.0 0 No Yes

250 Crude B to UV 136 12.0 1 Yes Yes

251 Crude B to UV 136 20.0 0 No Yes

252 Crude C to UV 137 12.0 1 Yes Yes

253 Crude C to UV 137 20.0 0 No Yes


































284 TFA to Pur Cols 188 6.0 0 No Yes

285 TFA to Pur Cols 188 25.0 0 Yes Yes

286 Wait 259 15.0 0 No Yes


288 TFA to PurCol A 189 1.0 999 No Yes

289 TFA to PurCol B 190 1.0 999 No Yes

290 TFA to PurCol C 191 1.0 999 No Yes

291 Wait 259 15.0 0 No Yes






297 Pur Vent 220 2.0 0 No Yes

298 Wait 259 23.0 0 No Yes





















319 Set Rmp Fxn Sns 265 0.0 10 No Yes

320 Set Rmp Fxn Trp 266 0.0 5 No Yes

321 Set Rmp Fxn Chk 267 0.0 5 No Yes

322 Set Rmp Fxn Dly 268 0.0 60 No Yes









328 H2O to UV 214 3.0 0 No Yes


































The Purification Biotin Cycle

Listing of BiotinCycle

Table D-3 Pur v4.40 Biotin Cycle



2 Pur Owns Depro 285 0.0 0 No Yes


4 Else 280 0.0 0 No Yes

5 Wait 259 60.0 0 No Yes

6 Wait 259 60.0 0 No Yes

7 Wait 259 60.0 0 No Yes










17 Pur Jaw Close 236 0.0 0 No Yes


19 Else 280 0.0 0 No Yes

20 Revive Act Cols 319 0.0 0 No Yes

21 Go Sub 287 0.0 11 No Yes




25 Go Sub 287 0.0 2 No Yes

26 Go Sub 287 0.0 1 No Yes

27 Go Sub 287 0.0 12 No Yes



30 Go Sub A 292 0.0 6 No Yes


32 Go Sub 287 0.0 2 No Yes

33 Go Sub 287 0.0 1 No Yes

34 Go Sub 287 0.0 12 No Yes


36 Go Sub B 293 0.0 6 No Yes



38 Go Sub 287 0.0 2 No Yes

39 Go Sub 287 0.0 1 No Yes

40 Go Sub 287 0.0 12 No Yes


42 Go Sub C 294 0.0 6 No Yes


44 Go Sub 287 0.0 2 No Yes

45 Go Sub 287 0.0 12 No Yes











56 Go Sub 287 0.0 3 No Yes

57 Go Sub 287 0.0 8 No Yes

58 Go Sub 287 0.0 3 No Yes



61 Go Sub 287 0.0 5 No Yes

62 Go Sub 287 0.0 3 No Yes

63 Go Sub 287 0.0 5 No Yes

64 Go Sub 287 0.0 3 No Yes

65 Go Sub 287 0.0 4 No Yes

66 Go Sub 287 0.0 3 No Yes

67 Go Sub 287 0.0 4 No Yes

68 Go Sub 287 0.0 3 No Yes

69 Go Sub 287 0.0 9 No Yes

70 Go Sub 287 0.0 9 No Yes

71 Go Sub 287 0.0 5 No Yes

72 Go Sub 287 0.0 3 No Yes

73 Go Sub 287 0.0 5 No Yes

74 Go Sub 287 0.0 3 No Yes




78 H2O to PurCol A 184 5.0 0 No Yes




79 H2O to PurCol B 185 5.0 0 No Yes

80 H2O to PurCol C 186 5.0 0 No Yes


82 Go Sub 287 0.0 3 No Yes


84 Go Sub 287 0.0 4 No Yes

85 Go Sub 287 0.0 3 No Yes


87 TEAA to Waste 182 5.0 0 No Yes

88 TEAA to PurCols 178 25.0 0 No Yes

89 TEAA to PurCols 178 45.0 0 Yes Yes














103 Go Sub 287 0.0 10 No Yes

104 Endif 281 0.0 0 No Yes


106 Go Sub C 294 0.0 2 No Yes

107 Go Sub 287 0.0 11 No Yes







114 Xfer Coil C 168 12.0 1 Yes No

115 Xfer Coil C 168 20.0 0 No No

116 Go Sub B 293 0.0 2 No Yes

117 Xfer Coil B 167 12.0 1 Yes No

118 Xfer Coil B 167 20.0 0 No No

119 Go Sub A 292 0.0 2 No Yes




120 Xfer Coil A 166 12.0 1 Yes No

121 Xfer Coil A 166 20.0 0 No No



124 Go Sub 287 0.0 10 No Yes








132 AcAcid to Waste 202 2.0 0 No Yes

133 AcAcid to Purs 198 3.0 0 No Yes

134 AcAcid to Purs 198 30.0 0 Yes Yes






140 Go Sub A 292 0.0 7 No Yes

141 Go Sub B 293 0.0 7 No Yes

142 Go Sub C 294 0.0 7 No Yes

143 Go Sub 287 0.0 4 No No

144 Go Sub 287 0.0 3 No Yes

145 Go Sub 287 0.0 4 No Yes

146 Go Sub 287 0.0 3 No Yes


148 Go Sub 287 0.0 9 No Yes


150 Begin Loop 273 0.0 3 No No

151 Go Sub 287 0.0 4 No No

152 Go Sub 287 0.0 3 No Yes

153 End Loop 274 0.0 0 No No

154 20%ACN to Waste 197 4.0 0 No Yes

155 20%ACN to Purs 193 6.0 0 No Yes

156 20%ACN to Purs 193 15.0 0 Yes Yes

157 Go Sub 287 0.0 10 No Yes







161 Endif 281 0.0 0 No Yes



164 Go Sub A 292 0.0 1 No Yes

165 Go Sub A 292 0.0 12 No Yes




169 Pur A to UV 211 10.0 3 Yes Yes

170 Pur A to UV 211 20.0 0 No Yes

171 Go Sub A 292 0.0 14 No Yes

172 Go Sub A 292 0.0 15 No Yes

173 Go Sub B 293 0.0 1 No Yes

174 Go Sub B 293 0.0 12 No Yes

175 SC Next 244 0.0 0 No Yes


177 Pur B to UV 212 10.0 3 Yes Yes

178 Pur B to UV 212 20.0 0 No Yes

179 Go Sub B 293 0.0 14 No Yes

180 Go Sub B 293 0.0 16 No Yes

181 Go Sub C 294 0.0 1 No Yes

182 Go Sub C 294 0.0 12 No Yes

183 SC Next 244 0.0 0 No Yes


185 Pur C to UV 213 10.0 3 Yes Yes

186 Pur C to UV 213 20.0 0 No Yes

187 Go Sub C 294 0.0 14 No Yes

188 Go Sub C 294 0.0 17 No Yes

189 Go Sub 287 0.0 12 No Yes

190 SC Next 244 0.0 0 No Yes

191 SC Open 245 0.0 0 No Yes





196 Go Sub 287 0.0 3 No Yes

197 Go Sub 287 0.0 8 No Yes

198 Go Sub 287 0.0 3 No Yes










205 H2O to UV 214 5.0 0 No Yes


207 Wait 259 10.0 0 No Yes



210 H2O to UV 214 1.0 0 No Yes












222 DepLowBlk Flush 153 2.0 0 No No

223 Return Sub 288 0.0 0 No No































251 Crude A to UV 135 12.0 1 Yes Yes

252 Crude A to UV 135 20.0 0 No Yes

253 Crude B to UV 136 12.0 1 Yes Yes

254 Crude B to UV 136 20.0 0 No Yes

255 Crude C to UV 137 12.0 1 Yes Yes

256 Crude C to UV 137 20.0 0 No Yes


































287 TFA to Pur Cols 188 6.0 0 No Yes

288 TFA to Pur Cols 188 25.0 0 Yes Yes

289 Wait 259 15.0 0 No Yes


291 TFA to PurCol A 189 1.0 999 No Yes

292 TFA to PurCol B 190 1.0 999 No Yes

293 TFA to PurCol C 191 1.0 999 No Yes

294 Wait 259 15.0 0 No Yes






300 Pur Vent 220 2.0 0 No Yes

301 Wait 259 23.0 0 No Yes





















322 Set Rmp Fxn Sns 265 0.0 10 No Yes

323 Set Rmp Fxn Trp 266 0.0 5 No Yes

324 Set Rmp Fxn Chk 267 0.0 5 No Yes




325 Set Rmp Fxn Dly 268 0.0 60 No Yes






331 H2O to UV 214 3.0 0 No Yes

































Instrument Plumbing Diagram E-1

Instrument Plumbing Diagram E E

Limited Warranty Statement F-1

Limited Warranty Statement F

Applied Biosystems warrants to the customer that, for a period ending on the earlier of two (2) years from the completion of installation or twenty-seven (27) months from the date of shipment to the customer (the “Warranty Period”), the ABI 3948 Nucleic Acid Synthesis and Purification System purchased by the customer (the “Instrument”) will be free from defects in material and workmanship, and will perform in accordance with the specifications set forth in the ABI 3948 System Data Sheet (the “Specifications”).

During the Warranty Period, if the Instrument's hardware becomes damaged or contaminated or if the Instrument otherwise fails to meet the Specifications, Applied Biosystems will repair or replace the Instrument so that it meets the Specifications, at Applied Biosystems expense. However, if the Instrument's valves or reagent lines become damaged or contaminated, or if the chemical performance of the Instrument otherwise deteriorates due to solvents and/or reagents other than those supplied or expressly recommended by Applied Biosystems, Applied Biosystems will return the Instrument to Specification at the customer’s request and at the customer's expense. After this service is performed, coverage of the parts repaired or replaced will be restored thereafter for the remainder of the Warranty Period.

This Warranty does not extend to any Instrument or part which has been (a) the subject of an accident, misuse, or neglect, (b) modified or repaired by a party other than Applied Biosystems, or (c) used in a manner not in accordance with the instructions contained in the Reference Manual or the Instrument User's Manual. This Warranty does not cover the customer-installable accessories or customer-installable consumable parts for the Instrument that are listed in the Reference Manual or Instrument User's Manual. Those items may have their own separate warranties.

Applied Biosystems obligation under this Warranty is limited to repairs or replacements that Applied Biosystems deems necessary to correct those failures by the Instrument to meet the Specifications of which Applied Biosystems is notified prior to expiration of the Warranty Period. All repairs and replacements under this Warranty will be performed by Applied Biosystems on site at the Customer's location at Applied Biosystems sole expense.

No agent, employee, or representative of Applied Biosystems has any authority to bind Applied Biosystems to any affirmation, representation, or warranty concerning the Instrument that is not contained in Applied Biosystemss printed product literature or this Warranty Statement. Any such affirmation. representation or warranty made by any agent, employee, or representative of Applied Biosystems will not be binding on Applied Biosystems.

Applied Biosystems shall not be liable for any incidental, special, or consequential loss, damage or expense directly or indirectly arising from the purchase or use of the Instrument. Applied Biosystems makes no warranty whatsoever with regard to

F

F-2 Limited Warranty Statement

products or parts furnished by third parties; such products or parts will be subject to the warranties, if any, of their respective manufacturers.

This Warranty is limited to the original site of installation and is not transferable.

THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES, INCLUDING, WITHOUT LIMITATION, ANY IMPL!ED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS.

Index-1

Numerics3' phosphorus 3-34X12 configuration rack

standard white 2-22

AABI 3948

calculation of concentration 3-25

computer controlled 2-6Abort

defined 1-4Abort command

definition 4-21procedure 4-22

absorbancereported in ODUs (Optical Density

Units) and picomoles 2-5acetonitrile

wash before detritylation 3-9Alternative Chemistries 3-28aminomethyl linker

attached to support material 3-4ammonium hydroxide, use in

cleavage 3-16application memory setting

default 4-10Applications

used to create Multi-Order files 4-41

Apply Button 6-14Automation

Chemistry Processes 2-4Auto-res OK 0.01 PSI

parameter 4-28Auto-Resume feature

Instrument Preferences commandauto-resume feature 4-27

Auto-resume Minutes parameter 4-28

BB+ Tet Calibration

contents of view 6-8base deprotection

guidelines 3-17Baseline absorbance

setting by the system 3-24Beer’s Law 3-22Begin and End Procedures

defined 1-4

benzoylprotection group 3-7

Block functionsgas flush, pressurization, and

rinse A-22priming delivery lines A-21priming for Cleavage

Columns A-21priming for Purification

Columns A-22Bottle change functions A-20bottle procedure

editing 6-22

CCalculation

of concentration (pmol/µL) 3-25Capping

characteristics 3-13process 3-13

capping 3-13–??, 3-13–3-14by acetylation 3-13highly recommended 3-13needed because coupling is not

always quantitative 3-13reactive chains not affected by

capping 3-13Change Name command

definition 4-21procedure 4-26

Change Passwords commanddefinition 4-21

Check Illegal Values parameter 4-29chemical delivery system

pressure driven 2-3Chemistries

alternative 3-28Chemistry

of choice 3-7progress during a run 5-12solid phase synthesis 3-3

chemistryprogress of 5-12

Chemistry Panesdescription of 5-12

Chemistry Processesautomation 2-4First Four 2-4

Chemistry protocolsdescription C-2

Chemistry reactionsdetails of four reactions 3-6

Chemistry Stages A-3Cleavage

module 2-4process of 2-15

cleavage and deprotection 3-16–3-17

removal of protecting groups 3-17

Cleavage Cycle parameter 5-9Cleavage functions

delivery to cleavage columns A-17

gas flushes and pressurization A-17

oligonucleotide transfer A-18water delivery to deprotection

coil A-18cleavage jaw sensors 5-16Cleavage module

components 2-7location and description 2-15

Cleavage reaction 3-16Close All Synth Orders

command 4-3definition 4-3

Close command 4-3Coil Cool Secs (261) parameter 4-28collection needle

waste bottles 2-22Column turntable

purpose of 2-7Command-Key commands 4-4Command-key equivalents (hot keys)

Edit menu 4-17File menu 4-4

Commands and capabilitiesoverview 4-1

Communication Viewinitial database window 5-6

concentrated aqueous ammonium hydroxide

used to cleave the oligonucleotide 2-4

controlleralso fills out Synthesis

Orders 2-3real time control 2-3

Copy from Button 6-14Coupling

characteristics 3-12

Index

Index-2

coupling 3-11–3-12, ??–3-12effect of excess of

phosphoramidite 3-12mixed sequence probes 3-12Nucleophilic attack by

tetrazole 3-12reactivity order for cyanoethyl

phosphoramidites 3-12Creating

Synthesis Orders 4-38Creating a User Function

procedure 6-5Criteria

for converting ODU to mass or concentration 3-22

Critical messages C-42crude oligonucleotides

with the 5´ trityl group either on or off 2-4

Current date and timewhere displayed 5-15

cuvette 2-20cyanoethyl

protecting group 3-8Cyanoethyl phosphoramidite

four functional groups 3-8cyanoethyl phosphoramidites

reactivity order 3-12cycle

definition of A-3Cycle and Procedure Pop-Up Menus

Edit views 6-13

Ddenaturing media 3-20Deprotect Mins (169)

parameter 4-28Deprotect Temp (170)

parameter 4-28Deprotection

module 2-4process of 2-17time and temperature 2-4

deprotectionbase 3-17phosphate 3-16

deprotection coil sensors 5-17Deprotection module

components 2-7, 2-16location 2-16

Depurinationpurines susceptable to 3-9

depurinationminimization of 3-10

Detritylationchemistry process 3-9

diisopropylamino functional group 3-8

dimethoxytrityl groupremoval 3-5

DNA productsdescription of elution 2-4

DNA purification 3-20DNA storage

period of stability 3-27DNA Synthesis Cycle

four steps 3-3

EEdit Begin Procedure View 6-20Edit Bottle Procedure View 6-22

purpose of view 6-22Edit Cleavage Cycle View

purpose of view 6-18Edit commands

Find Same 4-18Read Selection 4-17Replace command 4-18

Edit Cycle and Procedure interfaceButtons 6-14entry fields 6-16interface elements 6-12

Edit End Procedure Viewediting 6-21

Edit Function Viewscrollable lists 6-4

Edit menu ??–4-19list of commands 4-17

Edit Purification Cycle Viewpurpose of view 6-19

edit sequence 4-18Edit Synthesis Cycle View

purpose of 6-17Edit Views, all

same general layout 6-12Electromechanical and electrical

drivers 2-6embedded software 2-6End Row on Jaw Leak

parameter 4-29End sequence

with A, B, C or T on the 3´ end 4-7

entering sequencesas mixed bases 4-39

Entry fieldsEdit Cycle and Procedure

interface 6-16Execute Button 6-14Exercising Manual Control

of a FunctionManual Control View

exercising control of afunction 6-4

exocyclic amines 3-5Export command 4-15export files

names and icons 4-15Export Procedure icons 4-15

Export Sequence commanddefinition 4-3

Ext. Coefficient 5, 6, 7, and 8description 4-28

Extinction coefficientmajor contributors to 3-22

Extinction coefficientsfor fluorescent dye labels 3-26

Ffailure sequences 3-13File commands

Import/Export 4-15Open 4-8Save a Copy In 4-13Send Copy to Synthesizer 4-14Send to Synthesizer 4-13

File menu 4-3–??list of commands 4-3list of tasks 4-3

final detritylationa trityl flush 3-9

Find command 4-18Find Same command

procedure 4-18Find/Find Same commands

definition 4-17Flow path

ACN to Column A (illustration) A-9

flowcell1 cm pathlength quartz 3-23

Function names A-7functional groups

on phosphoramidites 3-8Functions

three fundamental types A-7Time and Misc field entries A-7tracing flow paths A-8

functionsprinting 6-5

Functions in cyclesdescription C-2

GGas pressure sensors/sensor

drivers 2-6General activity messages C-40Generate Run File command 4-16

definition 4-3Generating Synthesis Orders

from Multi-Order files 4-42

HHardware

types of 2-7Hierarchical messages C-43high throughput 2-4

Index-3

IImport command 4-15Import Sequence command

definition 4-3Import/Export

discussion 4-15Importing and Exporting Sequences

Synthesis Orders 4-39Importing Synthesis Orders

process 5-21initial Chemistry Pane view 5-12Insert Button 6-15instrument control

real time 2-3Instrument Dip Switches

Instrument PreferencesInst rument Dip Swi tches

parameters 4-29Instrument Messages C-40Instrument plumbing diagram E-1Instrument Preferences command

definition 4-21purpose of 4-27Setup Variables 4-28

Instrument Test Viewactivating tests 6-8tests performed 6-7

Interruptdefined 1-4

Interrupt command 4-22definition 4-21

iodine reagentin oxidation 3-15

isobutyrylprotection group 3-7

JJaw Leak Test

in PSI parameter 4-28Jaw mechanism

illustration and detailed description 2-11

purpose of 2-7

Kkey terms

table of 1-4

LLabel View

information in individual labels 4-36

Label View informationin the five columns 4-36

Leak OK 0.01 PSIparameter 4-28

Limited warranty statement F-1Liquid sensors 2-6

conventions 5-16

list of open windows 4-30Log Dry Sensor Fxns

parameter 4-29Low pressure mercury lamp 3-23low-pressure mercury lamp

(P/N) 2-20

MMan Cont Jaw Testing

parameter 4-29Manual Control

Creating a User Function 6-5exercises valve functions A-10printing functions 6-5

Manual Control Viewviewing a function valve list 6-4

manual volumes, twopurposes of 1-1

Maxam-Gilbert sequencingreference to 3-9

Method of synthesisPhosphoramidite 2-3

Microphone-only messages C-42Misc (Miscellaneous) Procedures View

purpose of view 6-23Miscellaneous hardware

functions A-24Miscellaneous Procedures C-37mixed base entry 4-39Mixed sequence probes

synthesis of 3-12Mnfg. Jaw Test Mode

parameter 4-29modify

sequence 4-18module 2-4Molar extinction coefficent 3-22Monitor Chemistry View

interface information 5-11sequence pane

description 5-13Status and System

Message 5-13three chemistry panes 5-12

Monitor Chemistry viewdescription of three chemistry

panes 5-12general description 5-11using information 5-14

Monitor Instrument Viewother

parameters/conditions 5-18

Monitor Run Viewgeneral information 5-19types of information

provided 5-19monitoring

valves 5-15monitoring chemistry 5-14

Monomersother than standard

phosphoramidite 3-28Multi-Order files

types of applications used to create 4-41

Nname

changing the synthesizer 4-26New Synthesizer Order command

definition 4-3NMI reagent

in capping 3-13No Flow To Open Jaws

parameter 4-29non-valve hardware functions

pressure regulating A-23nucleosides

phosphoramidite 3-7Nucleotide 3´-hydroxyl

attachment to support 3-4

OODU

as a measure of concentration 3-22

criteria for converting to mass or concentration 3-22

definition 3-22ODU as the Unit of Measure

definitions which apply 3-22ODU Measurement

optimum measurement criteria 3-22

oligonucleotide productiondescription of process 2-3

oligonucleotide quantitation 3-22oligonucleotide yield

automatic measurement 3-23oligonucleotides

methods of purification 3-20storage for later use 3-27types to avoid storing 3-27

OneStep columncontains the 3´-terminal

nucleoside 2-4illustration and detailed

description 2-9moves between three

modules 2-4three of four chemistry processes

occur within 2-4OneStep column turntable

illustration and detailed description 2-10

OneStep™ Columnfour types 2-7

OneStep™ column 2-3

Index-4

Open command 4-8definition 4-3

Open Synthesizer command 4-3definition 4-3

Opening an Oligo Labels window for printing

alternate way 4-35oxidation 3-15Oxidation after capping

importance of 3-15Oxidation reaction 3-15

PPAGE and HPLC

use for purification 3-20Page Setup command

definition 4-3Parallel and serial User Functions

use together A-13Pause After

defined 1-4Pause After Command

programming a pause 4-23Pause After command

definition 4-21Pause On Jaw Leak

parameter 4-29Pause On Sensor Fail

parameter 4-29Phosphate deprotection 3-16phosphoramidite method of

oligonucleotide synthesischaracteristics 3-7

Phosphoramidite method of synthesisoverview 3-6

phosphoramidite method of synthesis 2-3

phosphoramidite nucleosidesFigure 3-7

Phosphoramidite nucleotidesfunctional groups 3-8structures and molecular

weights 3-7phosphoramidite sensors 5-17phosphoramidites

function groups 3-8phosphoramidites and tetrazole

delivered simultaneouslyl during coupling 3-11

polystyreneused as solid support for DNA

synthesis 3-4Polystyrene support

highly cross-linked 3-3Power Fail View

purpose of 6-6Pressure regulating functions A-23Pressure Regulation and Control

(PRC) modulecharacteristics 2-12

Pressure Regulation and Control module (PRC)

purpose of 2-7Printing labels from RunFiles

alternate way 4-35information in individual

labels 4-36procedure

definition of A-3types of A-3

Proceduresbottle change (Manual

Dilution) C-36End C-34miscellaneous C-37phosphoramidite

autodilution C-36Start Up C-32

proceduresfor beginning synthesis C-32

protecting groupsremoval 3-17

protection groups 3-7Protocol

defined 1-4Protocol Name parameter 5-9Purification 2-4

characteristics of 3-20other methods 3-20process of 2-19

Purification and quantitation functionscollecting and quantitating

samples A-19delivery of reagents to purification

columns/UV detector A-18

rinsing and flushing deprotection columns A-19

Purification biotin cyclelisting D-48

purification coil sensors 5-17Purification Cycle parameter 5-10Purification dye cycle

listing D-39Purification module

components 2-7location and description 2-18

Purification stages 3-20purification step

omitting 3-5purified oligonucleotides

must be synthesized with the 5´ trityl group on 2-4

QQuantitation

general description 2-5Quantitation module 2-20

components 2-7

quantitation of oligonucleotidesby UV spectroscopy 3-22

RRacks

two types of 2-22racks

outside dimensions 2-22reaction column chamber

OneStep column 2-3reactive chains

still trityl blocked 3-13Read Selection command 4-17,

4-40definition 4-17procedure 4-17

red rackoptional 2-22

Reference manuallisting of chapter contents 1-2

Refrigerated storagein solution 3-27

Regular reports messages C-40Replace 4-18Replace command

procedure 4-18Replace/Replace Same commands

definition 4-17Reserved parameter 4-29Resume command 4-22

definition 4-21Run Protocol View 5-10

Cleavage Cycle parameter 5-9Protocol Name parameter 5-9Synthesis Cycle parameter 5-9used to create protocols 5-9

Run Setup Viewassigning chemistry 5-25autoSorting 5-25description of Open Dialog box

buttons 5-22information provided for a selected

sequence 5-24list of Menus and Buttons 5-8list of tasks performed

accessing 5-7selecting orders for the next

run 5-23RunFiles

list of types of information 4-32

SSafe/Sensor Buttons 6-15Sample collection

general description 2-5unattended collection of 48 DNA

samples 2-5

Index-5

Sample Collection modulecomponents 2-7components and

illustration 2-21needle assembly 2-21

Sample Labeling featureprocedure 4-33

Sample rackscharacteristics 2-22

sample vialssample volume 2-5

Save & Reopen command 4-3definition 4-3

Save a Copy In Commanddiscussion 4-13

Save a Copy In command 4-13definition 4-3

Save and Reopen commanddiscussion 4-14

Save As command 4-3definition 4-3

Save command 4-3definition 4-3

Scaling factor 3-24Select All command

definition 4-17Send Copy to Synthesizer

command 4-14definition 4-3

Send to Synthesizer command 4-3definition 4-3types of files sent 4-13

Sensor User Function namescomponents of A-12

Sensor-Controlled User Function example A-14

Sensorsgeneral characteristic A-11location of A-11names and locations A-13retries A-12use of parallel with serial Sensor

User Functions A-13septum sheet

purpose of 2-5where located 2-5

sequenceimport

export 4-15Sequence entry

Synthesis Orders 4-6sequence modification 4-18sequence pane description

Monitor Chemistry View 5-13Setup Choices

Instrument PreferencesSetup Choices 4-29

Setup Variables 4-28Show Clipboard command

definition 4-17

Solid supportcharacteristics 3-3

Solid–phase synthesis chemistry 3-3Special functions

overview of B-2Standard Cleavage/Deprotection Cycle

listing C-12summary C-9

Standard Purification Cyclelisting C-23summary C-18

Standard Synthesis Cyclesummary C-3

Standard Synthesis Cycle listing C-5Status and System Messages

Monitor Chemistry View 5-13Storage

frozen in 20% acetonitrile 3-27guidelines 3-27of crudes 3-27other liquid media 3-27

string substitutions 4-18succinate ester bond 3-4Support

characteristics 3-4support material

highly cross-linked polystyrene 3-4

Synchronize Clocks command 4-24definition 4-21

Synthesismodule 2-4process 3-3process of 2-14

synthesiscompletion of 3-5

Synthesis column functionsflushing and back flushing A-16

Synthesis Cycleconcluding steps 3-5

Synthesis Cycle parameter 5-9synthesis jaw sensors 5-16synthesis manifold sensors 5-17Synthesis Module

components 2-7Synthesis module

location and description 2-13Synthesis Order

sequence entry 4-39Synthesis Order command

description of fields 4-5types of information 4-5

Synthesis Order informationprovided for a selected

sequence 5-24

Synthesis Ordersactions after Sequence

Entry 4-40importing and exporting

sequences 4-39information contained

within 4-38ways of creating 4-38

Synthesis scaledescription C-2

Synthesis valve functions A-15Synthesis Window

editing 5-1Synthesizer Commands

list of definitions 4-21Synthesizer commands

Synchronize Clocks commandprocedure 4-24

Synthesizer menu 4-21–??Abort 4-22Change Name 4-26Interrupt 4-22Resume 4-22Synchronize clocks 4-24

Synthesizer Windowaccessing by password 5-6general information 5-4keyboard shortcuts 5-5list of views 5-4

Synthesizer windowlist of views 4-11

Synthesizer Window viewslist of 4-11

System Functions A-7System messages C-38

categories of messages C-39critical messages C-42hierarchical messages C-43message conventions C-39Microphone-only

messages C-42regular reports messages C-40types of messages C-38

TTCA

in detritylation 3-9three stations

concurrent use of A-4trichloroacetic acid 3-9

for DMT removal 3-5trityl

protecting group 3-8trityl group

as handle for purification 3-5Turntable control functions A-23turntable positions (A, B, C) 5-16types of runs

pre-programmed or custom 2-3Types of storage 2-6

Index-6

UUse Sounds command

definition 4-17User 4x12 Tube Rack

parameter 4-29User bulletins

contents and purpose 1-4User cycles

description C-2User Functions A-7

general description A-12User functions A-20UV detection system

description 2-20UV detector sensor 5-17UV flowcell

illustration 3-23UV hardware 3-23UV scaling factor 3-24UV spectroscopy 3-22UV-light source

254 nm 2-20

VValve Functions

creating your own A-10types of A-10uses of A-10

Valve functionslist of function categories A-15

valve statusoff or on 5-15

Valveslist of valves A-5

vial septumchemically inert 2-5

Viewing a function valve list 6-4

Wwaste bottles

location of bottles for collection needle rinse 2-22

wetted componentschemically inert 2-11

Window menu 4-30description 4-30

XXfer From Coil (261) parameter 4-28Xfer Into Coil (260) parameter 4-28

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Printed in the USA, 04/2011Part Number 4303111C

abiâ„¢ 3948 reference manual - life technologies

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