sequencing chemistry guide - applied biosystems | … · 96-well reaction plate column purification...

ABI PRISM® 3100 Genetic Analyzer

Sequencing Chemistry Guide

© Copyright 2001, Applied Biosystems

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

FOR LIMITED LICENSE INFORMATION, PLEASE SEE THE ABI PRISM® 3100 GENETIC ANALYZER USER’S MANUAL.

ABI, BigDye, CATALYST, Hi-Di, and POP-6 are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

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Contents

i

1 Manual OverviewChapter Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

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

About This Manual. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Who Should Use This Manual. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

If You Need More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Documentation User Attention Words. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Chemical Hazard Warning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Chemical Waste Hazard Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Site Preparation and Safety Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Instrument Safety Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Before Operating the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Ordering MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5

2 About the 3100 Genetic AnalyzerChapter Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

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

Instrument Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

3100 Genetic Analyzer Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Sequencing Chemistries Supported . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Run Cycle Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3

Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4

Capillary vs. Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4

Advantages of 3100 Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

3100 POP-6 Polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

Factors Affecting Instrument Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

Template Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

Template Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8

Compatible Plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10

Compatible Plates from Applied Biosystems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10

ii

3 BigDye Terminator ChemistryChapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

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

Section: Setting Up BigDye Terminator Reactions for a 96-Well Format . . . . . . . . .3-3In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

Preparing the Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Template Quantity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Two Cycle Sequencing Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5

Preparing 1X Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5

Preparing 2X Reactions for BACs, PACs, YACs, and Cosmids . . . . . . . . . . . . . . . . . . . 3-6

Preparing 2X Reactions for Bacterial Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6

Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7

Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7

Modifying the Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7

Cycle Sequencing Using Standard Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7

Cycle Sequencing for BACs, PACs, YACs, and Cosmids . . . . . . . . . . . . . . . . . . . . . . . 3-8

Cycle Sequencing for Bacterial Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8

Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

96-Well Plate Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

Isopropanol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10

Ethanol Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-11

Section: Setting Up BigDye Terminator Reactions for a 384-Well Format . . . . . . .3-13In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-13

Preparing the Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-14

Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-14

Template Quantity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-14

Volume Capacity Restrictions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-14

Two Cycle Sequencing Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15

Preparing Reactions for 384-Well Plate Column Purification . . . . . . . . . . . . . . . . . . . 3-15

Preparing Reactions for Alcohol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16

Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17

Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17

Modifying the Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17

Cycle Sequencing Using Standard Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17

Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18

384-Well Plate Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18

iii

Ethanol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19

Isopropanol Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20

4 BigDye Primer ChemistryChapter Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

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

Section: Setting Up BigDye Primer Reactions for a 96-Well Format . . . . . . . . . . . . 4-3In This Section. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3


Using Control DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Template Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Two Cycle Sequencing Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Preparing 1X Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5

Preparing 2X Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5


Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Cycle Sequencing Using Standard Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Cycle Sequencing for BAC DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7

Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Ethanol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Section: Setting Up BigDye Primer Reactions for a 384-Well Format . . . . . . . . . . 4-11In This Section. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11


This Procedure Is Not Recommended . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12

Using Control DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12

Template Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12

Two Cycle Sequencing Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12

Preparing 1X Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13

Preparing 0.5X Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13


Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Cycle Sequencing Using Standard Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14


Ethanol Precipitation Not Recommended . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-15

iv

5 dRhodamine Terminator ChemistryChapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1

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

Section: Setting Up dRhodamine Terminator Reactions for a 96-Well Format . . . .5-3In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3


Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4

Template Quantity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4

Preparing Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5


Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6

Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6

Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6


Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7

96-Well Reaction Plate Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7

Ethanol/Sodium Acetate Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-8

Section: Setting Up dRhodamine Terminator Reactions for a 384-Well Format . .5-11In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11


Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12

Template Quantity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12

Volume Capacity Restrictions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12

Preparing Reactions for 384-Well Plate Column Purification . . . . . . . . . . . . . . . . . . . 5-13

Preparing Reactions for Alcohol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-13


Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-14

Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-14

Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-14

Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15

384-Well Plate Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15

Ethanol/Sodium Acetate Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15

6 Sample InjectionsChapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

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

Preparing Samples for Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2

Injection Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2

Sample Volumes for Reaction Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2

v

Covering Sample Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2

Centrifuging Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3

For 96 and 384-Well Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3

Optimizing Electrokinetic Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4

Signal Too Strong . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4

Signal Too Weak Using 50-cm Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4

Signal Too Weak Using 36-cm Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5

Poor Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5

Optimizing Electrophoresis Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6

Run Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6

Run Temperature and Run Voltage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8

Laboratory Temperature and Humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9

For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9

Deionizing and Storing Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1

Formamide, Denaturation Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1

Option to Purchase or to Make . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1

Purchasing Hi-Di Formamide from Applied Biosystems . . . . . . . . . . . . . . . . . . . . . . . .A-1

The Problem with Commercial Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1

Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-3

Ion-Exchange Resin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-3

Calibrating the Conductivity Meter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-4

Preparing EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-4

Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-4

Using the Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-5

Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-1

Contacting Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-1

Hours for Telephone Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-1

To Contact Technical Support by Telephone or Fax . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-2

To Reach Technical Support Through the Internet . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-5

To Obtain Documents on Demand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-5

Chemistry Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-1

Troubleshooting Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-1

Index

Manual Overview 1-1

Manual Overview 1

Chapter Summary

In This Chapter The following topics are covered in this chapter:

Topic See Page

About This Manual 1-2

Safety 1-3

1

1-2 Manual Overview

About This Manual

Purpose This manual describes the chemistry protocols for the ABI PRISM® 3100 Genetic Analyzer. It is organized into task-oriented topics, each with a step-by-step description of how to perform the task.

Note This guide is specific to the 3100 Genetic Analyzer. For general chemistry information about kits, dyes, enzymes, protocols, and troubleshooting, please refer to the Automated DNA Sequencing Chemistry Guide (P/N 4305080).

Who Should UseThis Manual

This manual is designed for those persons preparing DNA samples for loading onto the 3100 Genetic Analyzer.

If You Need MoreInformation

If you need more information on... See the... Part Number

site preparation ABI PRISM 3100 Genetic Analyzer Site Preparation and Safety Guide

4315835

general chemistry Automated DNA Sequencing Chemistry Guide

4305080

an abbreviated description of how to sequence on the 3100 Genetic Analyzer

ABI PRISM 3100 Genetic Analyzer Quick Start Guide for Sequencing

4315831

an abbreviated description of how to perform fragment analysis on the 3100 Genetic Analyzer

ABI PRISM 3100 Genetic Analyzer Quick Start Guide for Fragment Analysis

4315832

Manual Overview 1-3

Safety

Documentation UserAttention Words

Five user attention words appear in the text of all Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below.

Note Calls attention to useful information.

IMPORTANT Indicates information that is necessary for proper instrument operation.

Cautions the user that a potentially hazardous situation could occur, causing injury to the user or damage to the instrument if this information is ignored.

Warns the user that serious physical injury or death to the user or other persons could result if these precautions are not taken.

Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury.

Chemical HazardWarning

CHEMICAL HAZARD. Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury, illness, or death.

� Read and understand the material safety data sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials.

� Minimize contact with and inhalation of chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS.

� Do not leave chemical containers open. Use only with adequate ventilation.

� Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer’s cleanup procedures as recommended on the MSDS.

� Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal.

Chemical WasteHazard Warning

CHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury, illness, or death.

� Read and understand the material safety data sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste.

� Handle chemical wastes in a fume hood.

� Minimize contact with and inhalation of chemical waste. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing).

� After emptying the waste container, seal it with the cap provided.

� Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations.

CAUTION!

WARNING!

DANGER!

WARNING!

WARNING!

1-4 Manual Overview

Site Preparation andSafety Guide

A site preparation and safety guide is a separate document sent to all customers who have purchased an Applied Biosystms instrument. Refer to the guide written for your instrument for information on site preparation, instrument safety, chemical safety, and waste profiles.

Instrument SafetyLabels

Safety labels are located on the instrument. Each safety label has three parts:

� A signal word panel, which implies a particular level of observation or action (e.g., CAUTION or WARNING). If a safety label encompasses multiple hazards, the signal word corresponding to the greatest hazard is used.

� A message panel, which explains the hazard and any user action required.

� A safety alert symbol, which indicates a potential personal safety hazard. See the ABI PRISM 3100 Genetic Analyzer Site Preparation and Safety Guide for an explanation of all the safety alert symbols provided in several languages.

Before Operating theInstrument

Ensure that everyone involved with the operation of the instrument has:

� Received instruction in general safety practices for laboratories

� Received instruction in specific safety practices for the instrument

� Read and understood all related MSDSs

Avoid using this instrument in a manner not specified by Applied Biosystems. Although the instrument has been designed to protect the user, this protection can be impaired if the instrument is used improperly.

CAUTION!

Manual Overview 1-5

Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below.

For chemicals not manufactured or distributed by Applied Biosystems, call the chemical manufacturer.

To order MSDSs... Then...

Over the Internet a. Go to our Web site at www.appliedbiosystems.com/techsupport.

b. Click MSDSs.

c. Enter keywords (or partial words), or a part number, or the MSDSs Documents on Demand index number.

d. Click Search.

e. Click the Adobe Acrobat symbol to view, print, or download the document, or check the box of the desired document and delivery method (fax or e-mail).

By automated telephone service from any country

Use “Documents on Demand” on B-5.

By telephone in the United States

Dial 1-800-327-3002, then press 1.

By telephone from Canada

By telephone from any other country

See “Regional Offices Sales and Service” on B-4.

To order in... Then dial 1-800-668-6913 and...

English Press 1, then 2, then 1 again

French Press 2, then 2, then 1

About the 3100 Genetic Analyzer 2-1

About the 3100 Genetic Analyzer 2

Chapter Summary


Topic See Page

Instrument Overview 2-2

Capillary Electrophoresis 2-4

Factors Affecting Instrument Performance 2-6

Compatible Plates 2-10

2

2-2 About the 3100 Genetic Analyzer

Instrument Overview

3100 GeneticAnalyzer

Description

The ABI PRISM® 3100 Genetic Analyzer is an automated, high-throughput, capillary electrophoresis system used for analyzing fluorescently labeled DNA fragments.

SequencingChemistries

Supported

The following chemistries are currently supported for use with the 3100 Genetic Analyzer:

� ABI PRISM® BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit v2.0

25,000 reactions (P/N 4314849)

5000 reactions (P/N 4314416)

1000 reactions (P/N 4314415)

100 reactions (P/N 4314414)

� ABI PRISM® BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit

5000 reactions (P/N 4303151)

1000 reactions (P/N 4303150)


� ABI PRISM® dGTP BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit


� ABI PRISM® BigDyeTM Primer Cycle Sequencing Ready Reaction Kit

5000 reactions with –21 M13 primer (P/N 403049)

5000 reactions with M13 reverse primer (P/N 403050)

100 reactions with –21 M13 primer (P/N 403051)

100 reactions with M13 reverse primer (P/N 403052)

� ABI PRISM® dRhodamine Terminator Cycle Sequencing Ready Reaction Kit

5000 reactions (P/N 4303143)




Run Cycle Overview

Step Event

1 DNA samples are prepared for sequencing in 96- or 384-well plates and placed on the autosampler of the instrument.

2 The capillaries are filled with 3100 POP-6 polymer, a medium that separates the DNA fragments.

3 The autosampler positions the 16 capillaries into the sample wells.

4 The fluorescently labeled DNA is loaded into the capillary by a short period of electrophoresis called electrokinetic injection. The capillaries are rinsed with water to remove sample adhering to the capillary sides.

5 The autosampler moves and positions the 16 capillaries into the buffer chamber for electrophoresis.

6 When the DNA fragments reach the detection window, the laser beam excites the dye molecules and causes them to fluoresce.

7 The fluorescence emissions from 16 samples are collected simultaneously and spectrally separated by a reflective spectrograph. The fluorescence emissions are focused as columns of light onto the charge-coupled device (CCD) that is part of the CCD camera.

8 The 3100 sequencing Data Collection software reads and interprets the fluorescence data, then displays the data as an electropherogram.


Capillary Electrophoresis

Capillary vs. GelElectrophoresis

Main Differences

Capillary and gel electrophoresis both separate DNA fragments by size through a sieving matrix in an electric field. There are three main differences between these separation methods:

� The structure of the sieving component

� The dissipation of heat

� The method of sample loading

The Sieving Component

Slab-gel electrophoresis, performed on the ABI PRISM® 373 and 377 DNA Sequencers, uses a gel created by the crosslinking of polyacrylamide and bis-acrylamide. The degree of crosslinking affects the porosity of the gel and thus the potential resolution of the DNA fragments. Once the gel matrix polymerizes, the gel is in a solid stationary phase.

Capillary electrophoresis is performed on the 3100 Genetic Analyzer, 3700 DNA Analyzer and the ABI PRISM® 310 Genetic Analyzer. The medium for capillary electrophoresis is a linear flowable polymer called 3100 POP-6™ polymer. These polymers can be pumped into and out of the capillaries for each new run.

Heat Dissipation

The method of heat dissipation for slab gel electrophoresis differs from that of capillary electrophoresis. During capillary electrophoresis heat is evenly and efficiently dissipated because there is more surface area in a capillary than in a gel plate. This enables higher voltages to be applied to migrating extension fragments without loss of resolution.

Sample Loading

Slab gels are loaded by inserting a pipet tip between two glass plates and into the pre-formed wells of the gel slab. The sample (including salts and proteins) is layered onto the top of the gel slab.

Samples for capillary electrophoresis are loaded into the capillary via electrokinetic injection. During electrokinetic injection negative ions such as extension fragments are injected into the capillary. However, no sample volume is injected or lost.


Advantages of 3100Capillary

Electrophoresis

The advantages of capillary electrophoresis on the 3100 Genetic Analyzer are:

� Rapid separation of DNA fragments.

� No gel pouring required. The 3100 Genetic Analyzer uses a liquid polymer, which is flowable and replenishable.

� No manual sample loading required.

� No gel tracking required.

� Increased sensitivity resulting from the simultaneous detection of 16 samples.

3100 POP-6 Polymer The 3100 POP-6 polymer (P/N 4316357) is a flowable polymer that is available for use on the 3100 Genetic Analyzer.


Factors Affecting Instrument Performance

Overview The quality and quantity of DNA in a reaction can affect the performance of the 3100 Genetic Analyzer.

Template Quality

The presence of residual salts, proteins, RNA, and detergents can interfere with capillary electrophoresis and electrokinetic injection. Your current template purification methods may have to be modified to remove residual salts, proteins, and detergents.

Template Quantity

The amount of DNA template used in a sequencing reaction can affect the quality of data. Excess template makes data appear “top heavy” with strong peaks at the beginning of the run that fade rapidly. Too little template or primer reduces the signal strength and peak height. In the worst case, the noise level increases so that bases cannot be called.

Template Quality When preparing DNA templates, it is critical to avoid the following:

� Residual salts

� Proteins

� Residual detergents

� Residual RNA

Effect of Residual Salts

The 3100 Genetic Analyzer is especially susceptible to salt in samples, either from template preparation or from cycle sequencing reactions. The negative ions in salts are preferentially injected into the capillaries during electrokinetic injection, leading to lower signal. Applied Biosystems scientists recommend implementing an efficient method to remove excess salts. Refer to the Automated DNA Sequencing Chemistry Guide for protocols.

Figure 2-1 shows the effect of salt during electrokinetic injection. The capillary marked “A” is a standard prepared using the recommended protocol; the capillaries marked “B” are standards with 10 mM NaCl added. The gel image in Figure 2-1 was artificially created by compressing the real-time data.

IMPORTANT When excess salts are present in the sample, negative ions compete and interfere with the injection of larger DNA extension fragments. This leads to shortened read lengths.


Figure 2-1 The effect of salt during electrokinetic injection

Effect of Proteins

Many DNA preparation methods for sequencing require the recovery of DNA from lysed bacterial cultures. Unless DNA is carefully purified, protein can remain in the DNA samples. Protein can be injected and adhere to the walls of the capillary, thus affecting data resolution and capillary lifetime.

Effect of Residual Detergents

Some methods of template preparation, such as the Thermomax method for M13 preparation, use detergents such as Triton X-100 to lyse the protein coat of phage particles. Other detergents, such as sodium dodecyl sulfate (SDS), are used in plasmid purification protocols to lyse bacterial cells. Small, negatively charged detergents may be preferentially injected over DNA during electrokinetic injection. If present at high levels, detergents such as Triton X-100 and SDS will impact the life of the capillary and the quality of the sequencing data.

Effect of Residual RNA

Residual RNA that is present in DNA template preps competes with the DNA for injection into the capillary array. Residual RNA has the same effect as excess salt, that is, decreased signal and shortened read lengths.


Template Quantity Excess Template in Your Sample

Loading too much template can potentially interfere with capillary flow, or shorten the life of capillaries. When DNA template is present in excess, it will behave as proteins would, and accumulate in the capillary. This affects data resolution.

Figure 2-2 on page 2-8 shows raw data from a BigDye terminator reaction containing excess template. The presence of excess template results in “top heavy” data and short reads. Electrokinetic injection enhances the effects of excess template by preferentially injecting shorter DNA fragments.

Figure 2-2 Raw data showing the effect of excess template in a BigDye terminator reaction

While Figure 2-2 shows raw data from a BigDye terminator reaction containing excess template, Figure 2-3 on page 2-9 shows analyzed data for this sample. The electropherogram in Figure 2-3 displays peaks that are clearly off-scale and have overall shortened read lengths. The presence of excess template in the reaction and the preferential electrokinetic injection of small DNA fragments cause this effect.


Figure 2-3 Electropherogram from the sample run in Figure 2-2


Compatible Plates

Introduction Samples must either be prepared in a plate or transferred to a plate before being placed on the autosampler. Make sure that the plate is securely placed on the autosampler.

Compatible Platesfrom Applied

Biosystems

Applied Biosystems supplies two types of plates (shown below) that are compatible with the 3100 Genetic Analyzer.

IMPORTANT Presently, these are the plates recommended for use on the 3100 Genetic Analyzer. Other plate types may be of different dimensions and thus affect instrument performance. Plates of varying tube depths may damage the array or the autosampler.

MicroAmp Optical 96-Well Reaction Plate

MicroAmp 384-Well Reaction Plate

BigDye Terminator Chemistry 3-1

BigDye Terminator Chemistry 3

Chapter Summary


Topic See Page

Setting Up BigDye Terminator Reactions for a 96-Well Format 3-3

Preparing the Reactions 3-4

Performing Cycle Sequencing 3-7

Preparing Extension Products for Electrophoresis 3-9





3

3-2 BigDye Terminator Chemistry


Section: Setting Up BigDye Terminator Reactions for a 96-Well Format

In This Section This protocol describes how to prepare BigDye™ terminator cycle sequencing reactions in MicroAmp® Optical 96-Well Reaction Plates.

Topic See Page






Preparing the Reactions

Using Control DNA It is strongly recommended that you include a control DNA template as one of the templates in a set of sequencing reactions. The results from the control can help determine whether failed reactions are the result of poor template quality or sequencing reaction failure.

We recommend M13mp18 as a single-stranded control and pGEM®-3Zf(+) as a double-stranded control. All Applied Biosystems DNA sequencing kits provide pGEM control DNA. All dye terminator cycle sequencing kits include a –21 M13 control primer.

Template Quantity The table below shows the template quantities for the BigDye terminator chemistry for a 1X cycle-sequencing run.

Template Template Quantity

PCR product:

100–200 bp 1–3 ng

200–500 bp 3–10 ng

500–1000 bp 5–20 ng

1000–2000 bp 10–40 ng

>2000 bp 40–100 ng

Single-stranded 50–100 ng

Double-stranded 200–500 ng

Cosmid, BAC 0.5–1.0 µg

Bacterial genomic DNA 2–3 µg


Two CycleSequencing Options

The flexibility of the BigDye terminator chemistry allows two options for cycle sequencing, as shown in the table below.

Preparing 1XReactions

To prepare 1X BigDye terminator reactions:

Reaction Type Template Cycle See...

1X � PCR product

� Plasmid

� M13

Standard “Preparing 1X Reactions” on page 3-5.

High-sensitivity (2X)

� Large DNA templates

� Bacterial genomic DNA

Modified “Preparing 2X Reactions for BACs, PACs, YACs, and Cosmids” on page 3-6.

or

“Preparing 2X Reactions for BACs, PACs, YACs, and Cosmids” on page 3-6.

Step Action

1 For each reaction, add the following reagents to a separate tube:

2 Mix well and spin briefly.

3 Continue with “Performing Cycle Sequencing” on page 3-7.

Reagent Quantity

Terminator Ready Reaction Mix 8.0 µL

Template –

single-stranded DNA 50–100 ng

double-stranded DNA 200–500 ng

PCR product DNA 1–100 ng(depending on size, see “Template

Quantity” on page 3-4).

Primer 3.2 pmol

Deionized water q.s.

Total volume 20 µL


Preparing 2XReactions for BACs,

PACs, YACs, andCosmids

To prepare high-sensitivity (2X) BigDye terminator reactions for BACs, PACs, YACs, and cosmids:

Preparing 2XReactions for

Bacterial GenomicDNA

To prepare high-sensitivity (2X) BigDye terminator reactions for bacterial genomic DNA:

a. Shearing the DNA by passing it several times through a 21-guage, 1-inch long needle can improve signals.

Step Action




Reagent Quantity

Terminator Ready Reaction Mix 16 µL

DNA Template 0.5–1.0 µg

Primer 5–10 pmol


Total volume 40 µL

Step Action




Reagent Quantity

Terminator Ready Reaction Mix 16 µL

Templatea 2–3 µg

Primer 6–13 pmol


Total Volume 40 µL


Performing Cycle Sequencing

Overview The following three protocols are used for BigDye terminator cycle sequencing reactions.

� Cycle Sequencing Using Standard Conditions

� Cycle Sequencing for BACs, PACs, YACs, and Cosmids

� Cycle Sequencing for Bacterial Genomic DNA

Thermal Cyclers These protocols have been optimized for all Applied Biosystems thermal cyclers:

� The ABI PRISM® CATALYST 800 Molecular Biology Lab Station

� The ABI PRISM® 877 Integrated Thermal Cycler

� The GeneAmp® PCR Systems 9600 and 9700 (in 9600 Emulation Mode).

IMPORTANT The protocols contained in this section should work for all of these instruments. If you use a thermal cycler not manufactured by Applied Biosystems, you may need to optimize thermal cycling conditions. Ramping time is very important. If the thermal ramping time is too fast (>1 °C/second), poor (noisy) data may result.

Modifying theProtocols

These protocols work for a variety of templates. However, the following modifications may be made:

� For short PCR products, a reduced numbers of cycles can be used (e.g., 20 cycles for a 300-bp or smaller fragment).

� If the Tm of a primer is >60 °C, the annealing step can be eliminated.

� If the Tm of a primer is <50 °C, increase the annealing time to 30 seconds or decrease the annealing temperature to 48 °C.

� For templates with high GC content (>70%), heat the tubes at 98 °C for 5 minutes before cycling to help denature the template.

Cycle SequencingUsing Standard

Conditions

To perform cycle sequencing under standard conditions:

Step Action

1 Place the tubes in a thermal cycler and set the volume to 20 µL.

2 Repeat the following for 25 cycles:

� Rapid thermal rampa to 96 °C

� 96 °C for 10 seconds

� Rapid thermal ramp to 50 °C



� 60 °C for 4 minutes

a. Rapid thermal ramp is 1 °C/second.

3 Rapid thermal ramp to 4 °C and hold until ready to purify.

4 Spin down the contents of the tubes in a microcentrifuge.

5 Continue with “Preparing Extension Products for Electrophoresis” on page 3-9.


Cycle Sequencing forBACs, PACs, YACs,

and Cosmids

To perform cycle sequencing for BACs, PACs, YACs, and cosmids:

Cycle Sequencing forBacterial Genomic

DNA

To perform cycle sequencing for bacterial genomic DNA:

Step Action


2 Heat the tubes at 95 °C for 5 minutes.

3 Repeat the following for 30 cycles:a

� Rapid thermal rampb to 95 °C


� Rapid thermal ramp to 50–55 °C (depending on template)

� 50–55 °C for 10 seconds



a. Increasing the number of cycles in step 3 increases signal.

b. Rapid thermal ramp is 1 °C/second.




Step Action


2 Heat the tubes at 95 °C for 5 minutes.

3 Repeat the following for 45 cycles:a

� Rapid thermal rampb to 95 °C


� Rapid thermal ramp to 50–55 °C (depending on template)




a. Increasing the number of cycles in step 3 increases signal.

b. Rapid thermal ramp is 1 °C/second.





Preparing Extension Products for Electrophoresis

Overview Unincorporated dye terminators must be completely removed before the samples can be analyzed by electrophoresis. Excess dye terminators in sequencing reactions obscure data in the early part of the sequence and can interfere with basecalling.

For an example of the effect of unincorporated dye terminators on sequencing data, refer to the Automated DNA Sequencing Chemistry Guide.

Methods Several methods for preparing extension products for electrophoresis are presented to offer a choice of reagents and processes. We recommend performing controlled reactions with each method to determine the one that works best for you.

� The spin column and 96-well reaction plate methods remove most or all excess terminators if performed correctly, but are more costly than precipitation methods.

IMPORTANT Precipitation methods are cheaper, but are more likely to leave unincorporated dye-labeled terminators that can obscure data at the beginning of the sequence. We recommend that you use anhydrous (100%) isopropanol or nondenatured ethanol. Refer to Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions User Bulletin (P/N 4304665).

IMPORTANT When using alcohol precipitation methods, a 70% isopropanol or 70% ethanol wash step is required. This removes residual salts and residual unincorporated dyes. If salts and unincorporated dyes are not removed from the sequencing reaction, they will compete with the extension fragments during electrokinetic injection and result in weak signals.

For BigDye terminator chemistries, the following methods are recommended:

96-Well PlateColumn Purification

For large-scale procedures, you can use the following commercially available 96-well purification plates:

� 96-Well Spin Columns, Gel Filtration Kit (Edge Biosystems, P/N 94880)

� ArrayIt (Telechem, P/N DTC-96-100)

� Centri-Sep™ 96 plate (Princeton Separations, P/N CS-961)

� Multiscreen 96-Well Filter Plates (Millipore, P/N MADYEKIT1)

Refer to the manufacturer’s instructions procedures.

Chemistry Recommended Methods See

BigDye Terminator

Purifying PCR Fragments by Ultrafiltration Automated DNA Sequencing Chemistry

Guide

96-Well Plate Column Purification 3-9

Isopropanol Precipitation 3-10

Ethanol Precipitation 3-11


IsopropanolPrecipitation

You will need the following reagents and equipment for this procedure:

� Variable speed table-top centrifuge with microtiter plate, capable of reaching at least 1400 × g

� MicroAmp strip caps or adhesive-backed aluminum foil tape (3M Scotch Tape 431 or 439)1

IMPORTANT 75% Isopropanol (2-propanol) or 100% isopropanol (anhydrous) at room temperature

Note This procedure does not use salt.

1. In the USA, contact 3M at (800) 364-3577 for your local 3M representative. Use of other tapes may result in leakage or contamination of the sample.

To perform isopropanol precipitation:

Step Action

1 Remove the 96-well reaction plate from the thermal cycler. Remove the caps.

2 Add one of the following:

� 80 µL of 75% isopropanol -or-

� 20 µL of deionized water and 60 µL of 100% isopropanol

The final isopropanol concentration should be 60 ± 5%.

CHEMICAL HAZARD. Isopropanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. It may cause central nervous system effects such as drowsiness, dizziness, and headache, etc. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

3 Seal the plate with strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage.

4 Invert the plate a few times to mix, or vortex.

5 Leave the plate at room temperature for 15 minutes to precipitate the extension products.

Note Precipitation times <15 minutes will result in the loss of very short extension products. Precipitation times >24 hours will increase the precipitation of unincorporated dye terminators.

6 Place the plate in a table-top centrifuge with plate adaptor and spin it at the maximum speed, which must be Š1400 × g but <3000 × g

� 1400–2000 × g 45 minutes

� 2000–3000 × g 30 minutes

IMPORTANT Proceed to the next step immediately. If not possible, then spin the plate for an additional 2 minutes immediately before performing the next step.

7 Without disturbing the precipitates, remove the adhesive tape and discard the supernatant by inverting the plate onto a paper towel. Remove as much supernatant as possible.

8 Rinse the pellet by adding 150 µL of 70% isopropanol to each well.

9 Seal the plate with adhesive tape and invert the plate a few times to mix.

WARNING!


EthanolPrecipitation

With ethanol precipitation, traces of unincorporated terminators may be seen at the beginning of the sequence data (up to base 40), but this is usually minimal. Some loss in the recovery of the smallest fragments may also be observed.

IMPORTANT Where 95% ethanol is recommended in precipitation protocols, purchase non-denatured ethanol at this concentration rather than absolute (100%) ethanol. Absolute ethanol absorbs water from the atmosphere, gradually decreasing its concentration. This can lead to inaccurate final concentrations of ethanol, which can affect some protocols.

You will need the following equipment and reagents for this procedure:

� Variable speed table-top centrifuge with microtiter plate tray, capable of reaching at least 1400 × g


IMPORTANT 95% Ethanol (ACS reagent grade), non-denatured


10 Place the plate in a table-top centrifuge and spin at 2000 × g for 10 minutes.

11 Remove the adhesive tape. Discard the wash onto a paper towel that is folded to the size of the plate.

12 Place the inverted plate with the towel into the table-top centrifuge and spin at 700 × g for 1 minute.

13 Remove the plate and discard the paper towel.

Note Pellets may or may not be visible. Vacuum drying of the samples is not necessary.

To perform isopropanol precipitation: (continued)

Step Action

2. In the USA contact 3M at (800) 364-3577 for your local 3M representative. Use of other tapes may result in leakage or contamination of the sample.

To perform ethanol precipitation:

Step Action

1 Remove the 96-well reaction plates from the thermal cycler. Remove the caps from each plate.

2 Add the following:

� 16 µL of deionized water

� 64 µL of non-denatured 95% ethanol (EtOH)

The final ethanol concentration should be 60 ± 3%.

CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin. Exposure may cause central nervous system depression and liver damage. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

3 Seal the plate with strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage.

WARNING!


4 Invert the plate a few times to mix.



6 Place the plate in a table-top centrifuge with plate adaptor and spin it at the maximum speed, which must be Š1400 × g but <3000 × g:

� 1400–2000 × g for 45 minutes

� 2000–3000 × g for 30 minutes

IMPORTANT Proceed to the next step immediately. If not possible, then spin the plate for 2 minutes more immediately before performing the next step.


8 Rinse the pellet by adding 150 µL of 70% ethanol to each well.

9 Seal the plates with adhesive tape and invert the plate a few times to mix.


11 Remove the adhesive tape and discard the wash onto a paper towel that is folded to the size of the plate.




To perform ethanol precipitation: (continued)

Step Action


Section: Setting Up BigDye Terminator Reactions for a 384-Well Format

In This Section This section describes how to prepare BigDye™ terminator cycle sequencing reactions in MicroAmp® Optical 384-Well Reaction Plates.

Topic See Page









Template Quantity The table below shows the template quantities for the BigDye terminator chemistry.

Volume CapacityRestrictions

IMPORTANT The wells in the 384-well plates have a maximum capacity of 35 µL. A portion of this capacity will be utilized during the post-reaction clean up step. For this reason, choose your reaction setup based on the method you will use to prepare extension products for electrophoresis.


PCR product:

100–200 bp 1–3 ng

200–500 bp 3–10 ng

500–1000 bp 5–20 ng

1000–2000 bp 10–40 ng

>2000 bp 40–100 ng



Cosmid, BAC Not recommended

Bacterial genomic DNA Not recommended

If you are purifying extension products using...

Then set up your reactions following this procedure ...

384-well plate column purification � “Preparing Reactions for 384-Well Plate Column Purification” on page 3-15.

� “Preparing Reactions for Alcohol Precipitation” on page 3-16.

� ethanol precipitation

� isopropanol precipitation

“Preparing Reactions for Alcohol Precipitation” on page 3-16a.

a. This protocol ensures that you will not exceed the volume capacity of the 384-well plate.



The flexibility of the BigDye terminator chemistry allows two options for cycle sequencing, as shown in the table below.

Preparing Reactionsfor 384-Well Plate

Column Purification

IMPORTANT Follow this reaction set up if extension products will be purified using 384-well plate column purification. If you are purifying extension products using alcohol precipitation, refer to “Preparing Reactions for Alcohol Precipitation” on page 3-16.

To prepare BigDye terminator reactions for 384-well plate column purification:

Post-Reaction Purification Method Template Cycle See...

384-Well Plate Column Purification

� PCR product

� Plasmid

� M13

Standard “Preparing Reactions for 384-Well Plate Column Purification” on page 3-15.

� 384-well plate column purification

� Ethanol precipitation

� Isopropanol precipitation

� PCR product

� Plasmid

� M13

Standard “Preparing Reactions for Alcohol Precipitation” on page 3-16.

Step Action




Reagent Quantity


Template –





Primer 3.2 pmol


Total volume 20 µL


Preparing Reactionsfor Alcohol

Precipitation

Follow this procedure if you are preparing BigDye terminator reactions and preparing the extension products for electrophoresis using any of the methods:


� Ethanol precipitation

� Isopropanol precipitation

Step Action




Reagent Quantity


Template –





Primer 3.2 pmol


Total volume 10 µL



Overview The following protocol is used for BigDye terminator cycle sequencing reactions.


Thermal Cycler These protocols have been optimized for the GeneAmp® PCR System 9700 (in 9600 Emulation Mode).

IMPORTANT If you use a thermal cycler not manufactured by Applied Biosystems, you may need to optimize thermal cycling conditions. Ramping time is very important. If the thermal ramping time is too fast (>1 °C/second), poor (noisy) data may result.

Modifying theProtocols

These protocols work for a variety of templates. However, the following modifications may be made:

� For short PCR products, a reduced numbers of cycles can be used (e.g., 20 cycles for a 300-bp or smaller fragment).

� If the Tm of a primer is >60 °C, the annealing step can be eliminated.

� If the Tm of a primer is <50 °C, increase the annealing time to 30 seconds or decrease the annealing temperature to 48 °C.

� For templates with high GC content (>70%), heat the tubes at 98 °C for 5 minutes before cycling to help denature the template.


Conditions

To perform cycle sequencing using standard conditions:

Step Action

1 Place the tubes in a thermal cycler and set the volume to 20 µL for 1X reactions or 10 µL for half-volume reactions.
















Methods Several methods for preparing extension products for electrophoresis are presented to offer a choice of reagents and processes. We recommend performing controlled reactions with each method to determine the one that works best for you.

� The spin column and 384-well reaction plate methods remove most or all excess terminators if performed correctly, but are more costly than precipitation methods.

IMPORTANT Precipitation methods are cheaper, but are more likely to leave unincorporated dye-labeled terminators that can obscure data at the beginning of the sequence. We recommend that you use anhydrous (100%) isopropanol or nondenatured ethanol. Refer to Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions User Bulletin (P/N 4304665).

IMPORTANT When using precipitation methods, a 70% isopropanol or 70% ethanol wash step is required. This removes residual salts and residual unincorporated dyes. If salts and unincorporated dyes are not removed from the sequencing reaction, they will compete with the extension fragments during electrokinetic injection and result in weak signals.

For BigDye terminator chemistries, the following methods are recommended:


For large-scale procedures, you can use the following commercially available 384-well reaction plate:


� 384 System I (Edge Biosystems, P/N 95674)

Refer to the manufacturer’s instructions for procedures.


BigDye Terminator

Purifying PCR Fragments by Ultrafiltration Automated DNA Sequencing Chemistry

Guide


Ethanol Precipitation 3-19

Isopropanol Precipitation 3-20



With ethanol precipitation, traces of unincorporated terminators may be seen at the beginning of the sequence data (up to base 40), but this is usually minimal. Some loss in the recovery of the smallest fragments may also be observed.

IMPORTANT Where 95% ethanol is recommended in precipitation protocols, purchase non-denatured ethanol at this concentration rather than absolute (100%) ethanol. Absolute ethanol absorbs water from the atmosphere, gradually decreasing its concentration. This can lead to inaccurate final concentrations of ethanol, which can affect some protocols.

You will need the following equipment and reagents for this procedure:


� Adhesive-backed aluminum foil tape (3M Scotch Tape 431 or 439)3

� 95% Ethanol (ACS reagent grade), non-denatured




Step Action

1 Remove the 384-well reaction plates from the thermal cycler. Remove the seal from each plate.

2 To the 10 µL reaction volume add the following:

� 18 µL of non-denatured 95% ethanol (EtOH)



3 Seal the plate with a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage.





� 1400–2000 × g for 45 minutes

� 2000–3000 × g for 30 minutes

IMPORTANT Proceed to the next step immediately. If not possible, then spin the plate for 2 minutes more immediately before performing the next step.

WARNING!


IsopropanolPrecipitation


� Variable speed table-top centrifuge with microtiter plate, capable of reaching at least 1400 × g


� 100% isopropanol (anhydrous) at room temperature











Step Action

4. In the USA, contact 3M at (800) 364-3577 for your local 3M representative. Use of other tapes may result in leakage or contamination of the sample.

To perform isopropanol precipitation:

Step Action

1 Remove the 384-well reaction plates from the thermal cycler. Remove the seal from each plate.

2 To the 10 µL reaction volume add the following:

� 15 µL of 100% isopropanol

The final isopropanol concentration should be 60 ± 5%.

CHEMICAL HAZARD. Isopropanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. It may cause central nervous system effects such as drowsiness, dizziness, and headache, etc. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

3 Seal the plate by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage.

4 Invert the plate a few times to mix, or vortex.

WARNING!




6 Place the plate in a table-top centrifuge with plate adaptor and spin it at the maximum speed, which must be Š1400 × g but <3000 × g

� 1400–2000 × g 45 minutes

� 2000–3000 × g 30 minutes

IMPORTANT Proceed to the next step immediately. If not possible, then spin the plate for an additional 2 minutes immediately before performing the next step.


8 Rinse the pellet by adding 35 µL of 70% isopropanol to each well.

9 Seal the plate with adhesive tape and invert the plate a few times to mix.


11 Remove the adhesive tape. Discard the wash onto a paper towel that is folded to the size of the plate.




To perform isopropanol precipitation: (continued)

Step Action

BigDye Primer Chemistry 4-1

BigDye Primer Chemistry 4

Chapter Summary


Topic See Page

Setting Up BigDye Primer Reactions for a 96-Well Format 4-3








4

4-2 BigDye Primer Chemistry


Section: Setting Up BigDye Primer Reactions for a 96-Well Format

In This Section This protocol describes how to prepare BigDye™ primer cycle sequencing reactions in MicroAmp® Optical 96-Well Reaction Plates.

Topic See Page








We recommend M13mp18 as a single-stranded control and pGEM®-3Zf(+) as a double-stranded control. All Applied Biosystems® DNA sequencing kits provide pGEM control DNA. All dye terminator cycle sequencing kits include a –21 M13 control primer.

Template Quantity The table below shows recommended template quantities for the BigDye primer chemistry.


The flexibility of the BigDye primer chemistry allows two options for cycle sequencing, as shown in the table below.

IMPORTANT Prepare separate tubes for each of the four reactions (A, C, G, and T).


PCR product:

100–200 bp 2–5 ng

200–500 bp 5–10 ng

500–1000 bp 10–20 ng

1000–2000 bp 20–50 ng

>2000 bp 50–150 ng



Cosmid, BAC 0.5–1.0 µg


Reaction Type Template Cycle

1X � PCR product

� Plasmid

� M13

Standard

High-sensitivity (2X) � Large DNA template containing –21 M13 and/or M13 Reverse priming site

Modified



To prepare 1X BigDye primer reactions:


To prepare high-sensitivity 2X BigDye primer reactions:

Step Action

1 Aliquot the following reagents into four PCR tubes:


Reagent A (µL) C (µL) G (µL) T (µL)

Ready Reaction Premix 4 4 4 4

DNA template (see “Template Quantity” on page 4-4).

1 1 1 1

Total volume 5 5 5 5

Step Action






2 2 2 2

Total Volume 10 10 10 10



Overview The following two protocols are used for BigDye primer cycle sequencing reactions:


� Cycle Sequencing for BAC DNA







Conditions


Step Action








� 70 °C for 1 minute







4 Rapid thermal ramp to 4 °C and hold until ready to pool and precipitate.



Cycle Sequencing forBAC DNA

To perform cycle sequencing for BAC DNA:

Step Action


2 Begin thermal cycling with the following parameters:




3 Repeat the following for 20b cycles:







b. Increasing the number of cycles in steps 3 and 4 increases signal.

4 Repeat the following for 15b cycles:









Overview Excess dye primers must be completely removed before the samples can be analyzed by electrophoresis.

Method For BigDye primer chemistry, the standard method is ethanol precipitation without the addition of salt. A 70% ethanol wash is optional.

IMPORTANT Use non-denatured 95% ethanol rather than absolute (100%) ethanol. Absolute ethanol absorbs water from the atmosphere, gradually decreasing its concentration. This can lead to inaccurate final concentrations of ethanol, which can affect some protocols







IMPORTANT Use non-denatured 95% ethanol rather than absolute (100%) ethanol. Absolute ethanol absorbs water from the atmosphere, gradually decreasing its concentration. This can lead to inaccurate final concentrations of ethanol, which can affect some protocols.



Step Action

1 Remove the four 96-well plates from the thermal cycler. Remove the caps from each plate.

2 Briefly centrifuge the plate to collect the samples at the bottom of the tubes.

3 Using a multichannel pipet, pool the entire samples volumes from each of the four plates (i.e. containing the dideoxy A, C, G, and T reactions).

4 Add 53 µL of 95% ethanol to the pooled reactions. Seal the plate with strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage.



6 Leave the plate on ice or at 4 °C for 15 minutes to precipitate the extension products.


WARNING!



� 1400–2000 × g for 45 minutes

� 2000–3000 × g for 30 minutes

IMPORTANT Proceed to the next step immediately. If not possible, then spin the tubes for 2 minutes more immediately before performing the next step.

8 Without disturbing the precipitates, remove the adhesive tape and discard the supernatant by inverting the plate onto a paper towel.





Step Action


Section: Setting Up BigDye Primer Reactions for a 384-Well Format

In This Section This protocol describes how to prepare BigDye™ primer cycle sequencing reactions in MicroAmp® Optical 384-Well Reaction Plates.

Topic See Page







This Procedure IsNot Recommended

IMPORTANT This procedure is not recommended for 384-well plate formats. Excess BigDye dye primers must be completely removed by ethanol precipitation prior to electrophoresis. However, since the capacity of the 384-well plate is 35 µL, it is not possible to pool and precipitate reactions in a 384-well plate. The samples would have to be pooled and precipitated in a 96-well plate. If you wish to do this, then follow the protocol described below.



Template Quantity The table below shows recommended template quantities for the BigDye primer chemistry.


The flexibility of the BigDye primer chemistry allows two options for cycle sequencing, as shown in the table below.

IMPORTANT Prepare separate tubes for each of the four reactions (A, C, G, and T).


PCR product:

100–200 bp 2–5 ng

200–500 bp 5–10 ng

500–1000 bp 10–20 ng

1000–2000 bp 20–50 ng

>2000 bp 50–150 ng





Reaction Type Template Cycle

1X � PCR product

� Plasmid

� M13

Standard

0.5X � PCR product

� Plasmid

� M13

Standard



To prepare 1X BigDye primer reactions:

Preparing 0.5XReactions

To prepare 0.5X BigDye primer reactions:

Step Action






1 1 1 1


Step Action

1 Dilute 5X Sequencing Buffer (400 mM Tris-HCl, 10 mM MgCl2, pH 9.0—P/N 4305605, 600 reactions; 4305603, 5400 reactions) with four parts deionized water to 1X for use in this procedure.

2 Dilute each Ready Reaction Premix (A, C, G, T) 1:1 with 1X Sequencing Buffer in a separate tube (e.g., 2 µL of A Mix and 2 µL of 1X Sequencing Buffer).

3 Aliquot the following reagents into four PCR tubes for each DNA template:



Diluted Ready Reaction Premix

4 4 4 4


1 1 1 1




Overview The Cycle Sequencing Using Standard Conditions protocol is used for BigDye primer cycle sequencing reactions.




Conditions


Step Action



















EthanolPrecipitation Not

Recommended

IMPORTANT This procedure is not recommended for 384-well plate formats. Because the capacity of the 384-well plate is 35 µL, it is not possible to pool and precipitate reactions in a 384-well plate. The samples would have to be pooled and precipitated in a 96-well plate. If you wish to do this, refer to “Preparing Extension Products for Electrophoresis” on page 4-8.

dRhodamine Terminator Chemistry 5-1

dRhodamine Terminator Chemistry 5

Chapter Summary


Topic See Page

Setting Up dRhodamine Terminator Reactions for a 96-Well Format 5-3








5

5-2 dRhodamine Terminator Chemistry


Section: Setting Up dRhodamine Terminator Reactions for a 96-Well Format

In This Section This protocol describes how to prepare dRhodamine terminator cycle sequencing reactions in MicroAmp® Optical 96-Well Reaction Plates.

Topic See Page









Template Quantity The table below shows recommended template quantities for dRhodamine terminator chemistry.


PCR product:

100–200 bp 1–3 ng

200–500 bp 3–10 ng

500–1000 bp 5–20 ng

1000–2000 bp 10–40 ng

>2000 bp 40–100 ng






Preparing Reactions To prepare dRhodamine terminator reactions:

Step Action




Reagent Quantity


Template –





Primer 3.2 pmol


Total Volume 20 µL



Overview The following protocol is used for dRhodamine terminator cycle sequencing reactions.






Cycle Sequencing To perform cycle sequencing:

Step Action

















Methods Two methods for preparing extension products for electrophoresis are presented to offer a choice of reagents and processes.


For dRhodamine terminator chemistries, the following methods are recommended:

96-Well ReactionPlate Column

Purification

For large-scale procedures, you can use the following commercially available 96-well purification plates:

� 96-Well Spin Columns, Gel Filtration Kit (Edge Biosystems, P/N 94880)


� Centri-Sep™ 96 plate (Princeton Separations, P/N CS-961)

� Multiscreen 96-Well Filter Plates (Millipore, P/N MADYEKIT1)

Refer to the manufacturer’s instructions procedures.


dRhodamine Terminators

96-Well Reaction Plate Column Purification 5-7

Ethanol/Sodium Acetate Precipitation 5-8


Ethanol/SodiumAcetate Precipitation




� 3 M Sodium acetate, pH 4.6 (P/N 400320)




To perform ethanol/sodium acetate precipitation:

Step Action

1 Remove the 96-well reaction plate from the thermal cycler. Remove the caps from each tube.


� 2.0 µL of 3 M sodium acetate (NaOAc), pH 4.6

� 50 µL of 95% ethanol (EtOH)

The final ethanol concentration should be 65%.


3 Seal the tubes with strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the tubes to prevent any leakage.





� 1400–2000 × g for 45 minutes

� 2000–3000 × g for 30 minutes




WARNING!








To perform ethanol/sodium acetate precipitation: (continued)

Step Action


Section: Setting Up dRhodamine Terminator Reactions for a 384-Well Format

In This Section This protocol describes how to prepare dRhodamine terminator cycle sequencing reactions in MicroAmp® Optical 384-Well Reaction Plates.

Topic See Page









Template Quantity The table below shows recommended template quantities for dRhodamine terminator chemistry.

Volume CapacityRestrictions

IMPORTANT The wells in the 384-well plates have a maximum capacity of 35 µL. A portion of this capacity will be utilized during the post-reaction clean up step. For this reason, choose your reaction setup based on the method you will use to prepare extension products for electrophoresis.


PCR product:

100–200 bp 1–3 ng

200–500 bp 3–10 ng

500–1000 bp 5–20 ng

1000–2000 bp 10–40 ng

>2000 bp 40–100 ng





If you are purifying extension products using...

Then set up your reactions following this procedure ...

384-well plate column purification � “Preparing Reactions for 384-Well Plate Column Purification” on page 5-13.

� “Preparing Reactions for Alcohol Precipitation” on page 5-13.

ethanol/sodium acetate precipitation “Preparing Reactions for Alcohol Precipitation” on page 5-13a.

a. This protocol ensures that you will not exceed the volume capacity of the 384-well plate.


Preparing Reactionsfor 384-Well Plate

Column Purification

Follow this reaction set up if extension products will be purified using 384-well plate column purification. If you are purifying extension products using alcohol precipitation, refer to “Preparing Reactions for Alcohol Precipitation” on page 5-13.

Preparing Reactionsfor Alcohol

Precipitation

Follow this procedure if you are preparing dRhodamine terminator reactions and preparing the extension products for electrophoresis using any of the methods:


� Ethanol/sodium acetate precipitation

Step Action




Reagent Quantity


Template –





Primer 3.2 pmol


Total Volume 20 µL

Step Action




Reagent Quantity


Template –





Primer 3.2 pmol


Total volume 10 µL



Overview The following protocol is used for dRhodamine terminator cycle sequencing reactions.



Cycle Sequencing To perform cycle sequencing:

Step Action

1 Place the tubes in a thermal cycler and set the volume to 20 µL for 1X reactions or 10 µL for half-volume reactions.














Overview Unincorporated dye terminators must be completely removed before the samples can be analyzed by electrophoresis. Excess dRhodamine terminators in sequencing reactions obscure data in the early part of the sequence and can interfere with basecalling.


Methods Two methods for preparing extension products for electrophoresis are presented to offer a choice of reagents and processes.


For dRhodamine terminator chemistries, the following methods are recommended:


For large-scale procedures, you can use the following commercially available 384-well reaction plate:


� 384 System I (Edge Biosystems, P/N 95674)

Refer to the manufacturer’s instructions for procedures.

Ethanol/SodiumAcetate Precipitation




� 3 M Sodium acetate, pH 4.6 (P/N 400320)




dRhodamine Terminators


Ethanol/Sodium Acetate Precipitation 5-15


To perform ethanol/sodium acetate precipitation:

Step Action

1 Remove the 384-well reaction plate from the thermal cycler. Remove the seal from each tube.



� 1.0 µL of 3 M sodium acetate (NaOAc), pH 4.6

� 18 µL of 95% ethanol (EtOH)



3 Seal the tubes by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the tubes to prevent any leakage.





� 1400–2000 × g for 45 minutes

� 2000–3000 × g for 30 minutes










To perform ethanol/sodium acetate precipitation: (continued)

Step Action

WARNING!

Sample Injections 6-1

Sample Injections 6

Chapter Summary


Topic See Page

Preparing Samples for Injection 6-2

Centrifuging Samples 6-3

Optimizing Electrokinetic Injection 6-4

Optimizing Electrophoresis Conditions 6-6

6

6-2 Sample Injections

Preparing Samples for Injection

Injection Solutions Before DNA samples and standards can be placed on the ABI PRISM® 3100 Genetic Analyzer, they must be resuspended in formamide.

IMPORTANT Covered samples can be stored in formamide for 24 hours at room temperature. Uncovered samples can only be stored in formamide for 8 hours.

CHEMICAL HAZARD. Formamide is harmful if absorbed through the skin and may cause irritation to the eyes, skin, and respiratory tract. It may cause damage to the central nervous system and the male and female reproductive systems, and is a possible birth defect hazard. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

Sample Volumes forReaction Plates

IMPORTANT Always stay within the volume range specified below.

For correct delivery of samples to the capillary tips, the sample volumes in the 96-well reaction plate wells must remain within the ranges specified in the table below.

For 96-Well Plates

For 384-Well Plates

Covering SamplePlates

Samples in injection solution are subject to evaporation or degradation depending on the injection solution used. The longer the samples are exposed to air, the more likely this problem will occur. To avoid issues with evaporation or degradation, Applied Biosystems recommends immediately covering the sample plates with the provided septa.

WARNING!

Reaction TypeType of Reaction Plate

Minimum Volume (µL)

Maximum Volume (µL)a

a. The maximum volume is to ensure that the septa does not touch the sample and cause cross-contamination.

Recommended Volume (µL)

1X 96-well 10.0 150 10–30

High-Sensitivity (2X)

96-well 10.0 150 10–30

Reaction TypeType of Reaction Plate

Minimum Volume (µL)

Maximum Volume (µL)a

a. The maximum volume is to ensure that the septa does not touch the sample and cause cross-contamination.

Recommended Volume (µL)

For 384-well column purification

384-well 10.0 15 10–15

For precipitation

384-well 10.0 15 10–15


Centrifuging Samples

For 96 and 384-WellPlates

Before placing your plates on the autosampler, you must centrifuge them to bring the samples down to the bottom of the tubes or wells. Failure to do this properly will result in the samples not being injected into the capillaries.

To centrifuge your plates:

Step Action

1 Seal the plates.

2 Centrifuge the 96- or 384-well reaction plates at 2000 x g for 1 minute.

3 Hold the plates up to the light and examine them carefully to make sure that every sample is positioned at the bottom of the tube or well. Before you place your plates on the plate deck, the samples in your tubes or wells should:

If one or more samples are not positioned correctly, repeat step 2.

4 Your samples are now ready to be placed on the plate deck.

Look like this... Not look like this... not look like this...

The sample is positioned correctly in the bottom of the tube or well.

The sample lies on the side wall because the plate or tube was not centrifuged.

An air bubble lies at the bottom of the tube or well because the sample was not:

� Centrifuged with enough force, or

� Centrifuged for enough time


Optimizing Electrokinetic Injection

Overview Optimizing electrokinetic injection can greatly improve data quality and run-to-run reproducibility. The goal is to inject sufficient DNA to yield peaks of adequate height (i.e., data with a good signal-to-noise ratio) while maintaining resolution and read length.

The 3100 Genetic Analyzer run modules have preset values for injection times and voltages. These values are adequate for most applications. However, you should consider modifying the injection parameters when:

� The signal is too strong

� The signal is too weak

� The resolution is poor.

IMPORTANT For information on setting electrokinetic injection values, refer to the ABI PRISM 3100 Genetic Analyzer User’s Manual.

Signal Too Strong If the signal is too strong:

� Decrease the injection time.

� Decrease the injection voltage.

� Decrease the concentration of DNA fragments in the sample.

Signal Too WeakUsing 50-cm Array

If the signal is too weak when using the 50-cm array:

� Increase the injection time. See the table below.

� Reduce the amount of salt in the sample.

� Increase the concentration of the DNA extension products.

� Do not increase the voltage. Increasing the voltage increases the signal, but may reduce resolution across the array. It is better to adjust the injection time in order to increase signal.

IMPORTANT Negative ions, e.g., EDTA and acetate, compete with DNA for injection. To reduce the amount of salt in a sequencing reaction, use column purification.

Injection Time

(seconds) Volts/cm

Total EKIa Product

(V-sec/cm)

a. electrokinetic injection (EKI)

Description

20 25 500 Default setting for injection time

40 25 1000 Maximum injection time recommended with minimal effect on resolution

80 25 2000 Signal strength increases at this injection time, but at the expense of resolution


Signal Too WeakUsing 36-cm Array

If the signal is too weak when using the 36-cm array:

� Increase the injection time. See the table below.

� Reduce the amount of salt in the sample.

� Increase the concentration of the DNA extension products.

� Do not increase the voltage. Increasing the voltage increases the signal, but may reduce resolution across the array. It is better to adjust the injection time in order to increase signal.

IMPORTANT Negative ions, e.g., EDTA and acetate, compete with DNA for injection. To reduce the amount of salt in a sequencing reaction, use column purification.

Poor Resolution If the resolution is poor:

� Decrease the injection time.

Decreasing the injection time will decrease the signal strength. To compensate for the loss in signal, lower the salt concentration in the sample or increase the DNA extension product concentration.

� Decrease the run voltage by approximately 15%.

Injection Time

(seconds) Volts/cm

Total EKIa Product

(V-sec/cm)

a. electrokinetic injection (EKI)

Description

15 32 480 Default setting for injection time

25 32 800 Maximum injection time recommended with minimal effect on resolution

50 32 1600 Signal strength increases at this injection time, but at the expense of resolution


Optimizing Electrophoresis Conditions

Overview Optimizing electrophoresis conditions (run time, run voltage, and run temperature) can greatly improve data quality, run-to-run reproducibility, and/or throughput. When selecting values for these parameters, consider the following factors:

� Read length desired

� Required degree of resolution

Run Time Determining Required Run Time

To ensure that you collect sufficient data to perform analysis, set the electrophoresis run time approximately 5–10 minutes higher than the migration time of the longest fragment you want to detect.

To change the data collection time refer to:

Changing Run Time for the 50-cm Array

You can change the data collection time for special requirements. For example, you can shorten the data collection time if you only need information about short extension products (e.g., in PCR sequencing).

Figure 6-1 illustrates the amount of time that elapses before a DNA fragment traveling through a 50-cm capillary reaches the fluorescence detector and data collection begins.

The graph assumes the following information:

� The run module is programmed for 41 minutes of protocol events, including 20 minutes of programmed delay time, before data collection starts.

� The separation voltage is 12,200volts.

� The separation distance is 50 cm with a total capillary length of 61 cm.

� The temperature is 50 °C.

Topic See Page

Changing Run Time for the 50-cm Array 6-6

Changing Run Time for the 36-cm Array 6-7


Figure 6-1 Graph of the time required for fragments to reach the detector using a 50-cm array

Changing Run Time for the 36-cm Array

You can change the data collection time for special requirements. For example, you can shorten the data collection time if you only need information about short extension products (e.g., in PCR sequencing).

Figure 6-2 illustrates the amount of time that elapses before a DNA fragment traveling through a 36-cm capillary reaches the fluorescence detector and data collection begins.

The graph assumes the following information:

� The run module is programmed for 26 minutes of protocol events, including 4 minutes of programmed delay time, before data collection starts.

� The separation voltage is 15,000 volts.

� The separation distance is 36 cm with a total capillary length of 47 cm.

� The temperature is 55 °C.

Fragment Size Collected (Basepairs)

Col

lect

ion

Tim

e (M

inut

es)


Figure 6-2 Graph of the time required for fragments to reach the detector using 36-cm array.

Run Temperatureand Run Voltage

Run Temperature

Protocols for sequencing applications with 3100 POP-6 specify a 50–55 °C electrophoresis temperature.

For templates that do not denature readily at 50 °C, the run temperature can be increased up to 60 °C.

Run Voltage

Decreased run voltage or temperature decreases the migration rate of fragments. Longer run times are required to collect the same size fragments as in standard conditions. The basecaller may not be able to analyze the spacing values.

Increased run voltage or temperature increases migration rates, allowing for shorter run times, but decreased resolution.

Col

lect

ion

Tim

e (M

inut

es)

Fragment Size Collected (Basepairs)


LaboratoryTemperature and

Humidity

The laboratory temperature should be maintained between 15 and 30 °C. It should not fluctuate more than ±6 °C during a run for optimal results.

The ABI PRISM 3100 Genetic Analyzer can tolerate up to 80% non-condensing relative humidity. Avoid placing the instrument near heaters, cooling ducts, windows, or back-to-back with another instrument.

For MoreInformation

For information on setting electrophoresis parameters, refer to the ABI PRISM 3100 Genetic Analyzer User’s Manual.

Purchasing or Preparing Formamide A-1

Purchasing or Preparing Formamide A

Deionizing and Storing Formamide

Formamide,Denaturation Agent

Formamide is used to denature the DNA samples before placing them on the ABI PRISM® 3100 Genetic Analyzer.

Option to Purchaseor to Make

There are two ways to obtain formamide for use with the 3100 Genetic Analyzer. You can:

� Prepare it yourself, using a mixed-bed (anionic and cationic) ion-exchange resin, as described in the directions below.

� Purchase Hi-Di Formamide from Applied Biosystems.

Purchasing Hi-DiFormamide from

Applied Biosystems

Hi-Di™ Formamide is suitable for use with the 3100 Genetic Analyzer. It is available from Applied Biosystems in 25-mL bottles (P/N 4311320).

The Problem withCommercialFormamide

Formamide purchased from commercial suppliers is often contaminated with variable amounts of water and undesirable organic and inorganic ions. In addition, formamide is often supplied in glass bottles, which, when opened, exposes the formamide to the air and allow it to absorb water.

Water reacts slowly with formamide to produce formic acid (methanoic acid) and ammonia. The ionic products of this reaction cause two problems:

� They compete significantly with the larger DNA ions for injection into the capillary, resulting in weaker signals.

� They react with the DNA, causing degradation of the sample.

Figure A-1shows the effect of formamide exposure to the air on electropherogram data.

A

A-2 Purchasing or Preparing Formamide

Figure A-1 The effect of exposing formamide-resuspended samples to air. The top panel shows electropherogram data from samples incubated for 51 hours with a lid. The bottom panel shows electropherogram data from samples incubated for 48 hours with no lid.


Materials Required The following materials are recommended for this procedure:

Ion-Exchange Resin The raw formamide is deionized with cationic and anionic mixed resins to remove impurities such as ammonium and formate ions. Deionization occurs at a slow mass- transfer rate in the equilibrium ion exchange kinetics due to:

� Physical changes in the resin in the presence of formamide

� Differences in molecular size and selectivity between the impurity ions and the H+ and OH- counterions.

Therefore, the conductivity of formamide must be monitored over time to determine the extent of deionization by the resin.

Material Description

Formamide The raw (prior to deionization) formamide should be:

� 99.5% purity or greater, with low water content

� Packed under an inert gas

� Have a conductivity of approximately 100 µSiemens/cm or less

Note Siemens, formerly called mho, are the units of measurement for specific conductance or conductivity.

Ion-exchange resin � Mixed-bed resin containing the following strong ion exchange functional groups:

R-SO3- (as H+ form) (cation)

R-CH2N+(CH3)3, (as OH- form) (anion)

– These groups are attached to a styrene divinylbenzene matrix with 8% cross-linkage.

� The minimum wet capacity is 1.5 meq/mL with 20–50 dry mesh size (AG501 X8, molecular biology grade mixed-bed resin)

� Available from Bio-Rad Laboratories (P/N 143-6424) or equivalent

Conductivity meter A commercial conductivity meter, or pH meter with an external conductivity cell, is sufficient to measure the conductivity of formamide if it has a cell constant of 1.0

Na2EDTA � Dihydrate (Mr 372.2)

� ACS reagent, 99% purity or greater

� Available from Sigma (P/N E4884) or equivalent

Container for storing formamide

Use a polypropylene screw-cap container

Note Glass containers are not recommended because of potential contamination from minerals.

A-4 Purchasing or Preparing Formamide

Calibrating theConductivity Meter

A conductivity meter and cell are needed to measure the effectiveness of the deionization process. The more deionized the formamide, the lower its conductivity.

Within the range or measurement, the conductivity meter should be routinely calibrated (to 50 µSiemens/cm or less). Calibrate the meter using standard solutions that are traceable to the National Institute of Standards and Technology (NIST). Because temperature affects conductivity, samples must be brought to room temperature before measuring the conductivity.

Preparing EDTA Alkaline EDTA (ethylenediaminetetraacetic acid) is added to the deionized formamide to stabilize it and to facilitate the electrokinetic injection of DNA. To minimize the amount of water added to the formamide, a concentrated (200-mM) stock solution of the EDTA is added.

Procedure CHEMICAL HAZARD. Formamide is harmful if absorbed through the skin and may cause irritation to the eyes, skin, and respiratory tract. It may cause damage to the central nervous system and the male and female reproductive system, and is a possible birth defect hazard. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

Note There is not a stopping point in this procedure. Complete the procedure from resin washing to freezing the formamide, without interruption.

To prepare the 200-mM EDTA stock solution:

Step Action

1 Add 7.44 g of Na2EDTA to 70 mL of deionized water and stir.

2 While stirring, slowly adjust to pH 8.0–8.8 by dropwise addition of a concentrated solution of sodium hydroxide.

CHEMICAL HAZARD. Sodium hydroxide (NaOH) causes severe burns to the skin, eyes, and respiratory tract. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

Note This helps the EDTA to dissolve over time, because the EDTA has a limited solubility until the pH is increased.

3 Dilute to 100 mL with deionized water.

4 Store at 4 °C.

WARNING!

To begin purification and measure conductivity:

Step Action

1 Calibrate the conductivity meter cell, and rinse the cell with distilled water.

2 In a polypropylene screw-cap container, wash 10 g of Bio-Rad AG501 X8 ion-exchange resin by swirling the sample with 10–20 mL of formamide for 1 minute.

3 Either decant off or filter through a course nylon or teflon filter, and discard the formamide.

4 Repeat steps 2 and 3 twice.

5 Add 100 mL of formamide to the washed resin.

6 Cap the mixture, ensuring that it is well sealed.

WARNING!


Using theFormamide

When ready for use, thaw and completely use one tube at a time before opening and exposing another. Store the tubes at 4 °C during the day for intermittent use. Otherwise, refreeze them. Minimize the number of freeze-thaw cycles for each tube.

7 Stir the mixture rapidly with a magnetic stirrer, or mix with an electric shaker, ensuring that the resin is suspended and mixes thoroughly with the formamide. Stir at room temperature for approximately 2 hours.

8 Stop stirring and allow the resin to settle for 5 minutes.

9 Remove a small aliquot of the mixture, and measure the conductivity at room temperature.

10 Rinse the conductivity cell with distilled water.

11

Note If the conductivity is not <5 µSiemens/cm after about 4.5 hours of mixing, repeat the entire procedure using a new lot of formamide and new resin.

Note Starting formamide with a higher purity and lower conductivity deionizes more efficiently.

To complete purification of deionized formamide:

Step Action

1 Vacuum-filter the deionized formamide using a 0.2- or 0.4-µm nylon or teflon filter.

2 Measure the final volume of deionized formamide.

3 Add the required volume of 200-mM EDTA to the deionized formamide to achieve a final concentration of approximately 0.3-mM EDTA.

Note After adding the EDTA, the final conductivity of the formulation is increased to approximately 25 µSiemens/cm. Use the equation below to calculate the volume of EDTA to add.

VEDTA (µL) = 1.5V Form(mL)

Where,

VEDTA(µL) = volume of EDTA to add in microliters

VFORM(mL)= measured volume of formamide in milliliters

Sample calculation with a final volume of 90-mL formamide:

VEDTA(µL) = 1.5 × 90 = 135 µL

4 Immediately aliquot the formamide into smaller polypropylene tubes and store at –15 to –20 °C for up to about 6 months.

To begin purification and measure conductivity: (continued)

Step Action

If the conductivity is... Then...

>5 µSiemens/cm Return to step 7, stirring for an additional 30 minutes.

<5 µSiemens/cm Continue with “To complete purification of deionized formamide:.”.

Technical Support B-1

Technical Support B

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You can contact Applied Biosystems for technical support by telephone or fax, by e-mail, or through the Internet. You can order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents 24 hours a day. In addition, you can download documents in PDF format from the Applied Biosystems Web site (please see the section “To Obtain Documents on Demand” following the telephone information below).

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B

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To contact Applied Biosystems Technical Support, use the telephone or fax numbers given below. (To open a service call for other support needs, or in case of an emergency, dial 1-800-831-6844 and press 1.)

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Troubleshooting C-1

Troubleshooting C

Chemistry Troubleshooting

TroubleshootingTable

Some common observations associated with the chemistries used on the ABI PRISM® 3100 Genetic Analyzer are listed below.

Observation Possible Cause Recommended Action

Poor data resolution Clogged capillary caused by an excess of protein, template, or other sample impurities

Replace the array.

Samples stored in formamide for longer than 24 hours at room temperature.

Re-prepare the samples.

Overloading of the sample Dilute the sample

Excess DNA injected into the capillary

Adjust the injection parameter. Refer to “Optimizing Electrokinetic Injection” on page 6-4.

Weak signal The quantity of template or primers in the sequencing reaction or the quantity of sample injected is too low.

Refer to this chemistry guide for a description of recommended template quantities.

If possible, resuspend the template in a smaller volume.

Increase injection time. Refer to “Optimizing Electrokinetic Injection” on page 6-4.

Excess salt is present in the sample.

Clean up the sample using a spin column or a 70% ethanol wash.

Samples stored in formamide for longer than 24 hours at room temperature.

Re-prepare the samples.

C

C-2 Troubleshooting

High background Dirty template Refer to the Automated DNA Sequencing Chemistry Guide for a description of how to clean up dirty templates.

Top-heavy samples Amount of template in the sequencing reaction is too high, creating an excess of short fragments that are preferentially injected into the capillary.

Refer to this chemistry guide for a description of recommended template quantities.

The concentration of extension products is too high.

Dilute the sample or decrease the injection time.

Blank lanes or no signal Blocked capillary caused by an excess of protein, template, or other impurities, or by dried polymer.

Clean up the DNA sample and repeat the cycle sequencing reaction.

Cycle sequencing reaction failed.

Breakdown of BigDye G nucleotide. Refer to “The Problem with Commercial Formamide” on page A-1.

Formamide degradation caused by exposure to the air.

Use formamide as recommended in Appendix A, “Purchasing or Preparing Formamide.”

Cover plates with septa.

Try using an alternative injection solution.

Observation Possible Cause Recommended Action

Index-1

Index

Numerics3100 Genetic Analyzer

capillary electrophoresis, optimizing 6-6 to 6-9chemistries supported 2-2overview 2-2 to 2-4performance, factors affecting 2-6plates, compatible 2-10run cycle overview 2-3

3100 POP-6 polymer, See POP-6 polymer36-cm array

changing run time 6-7optimizing electrophoresis 6-6

384-well formatBigDye primer reactions 4-11 to 4-15BigDye terminator reactions 3-13 to 3-21dRhodamine terminator reactions 5-11 to 5-16

384-well platecentrifuging samples 6-3

50-cm arraychanging run time 6-6optimizing electrokinetic injection 6-4optimizing electrophoresis 6-6

96-well formatBigDye primer reactions 4-3 to 4-9BigDye terminator reactions 3-3 to 3-12dRhodamine terminator reactions 5-3 to 5-9

96-well platecentrifuging samples 6-3recommended volume 6-2

BBacterial Artificial Chromosome (BAC), cycle

sequencing 3-8bacterial genomic DNA, cycle sequencing 3-8bases, compatible 2-10BigDye primer reactions

384-well format 4-11 to 4-150.5X reactions 4-131X reactions 4-13control DNA 4-12cycle sequencing 4-14ethanol precipitation 4-15extension products, preparing for

electrophoresis 4-15reaction dilutions 4-12reactions, preparing 4-12 to 4-13template quantity 4-12volume restrictions 4-12, 4-15

96-well format 4-3 to 4-91X reactions 4-52X reactions 4-5control DNA 4-4cycle sequencing for BAC DNA 4-7cycle sequencing, standard conditions 4-6

ethanol precipitation 4-8extension products, preparing for

electrophoresis 4-8reactions, preparing 4-4 to 4-5template quantity 4-4

BigDye terminator reactions384-well format 3-13 to 3-21

1X reactions 3-15control DNA 3-14cycle sequencing 3-17cycle sequencing options 3-15ethanol precipitation 3-19extension products, preparing for

electrophoresis 3-18 to 3-21isopropanol precipitation 3-20modifications to thermal cycling program 3-17preparing reactions for precipitation 3-16, 5-13reactions, preparing 3-14 to 3-16template quantity 3-14volume restrictions 3-14

96-well format 3-3 to 3-121X reactions 3-52X reactions for BACs, YACs, Cosmids 3-62X reactions for genomic DNA 3-6control DNA 3-4cycle sequencing 3-6cycle sequencing options 3-5ethanol precipitation 3-11extension products, preparing for

electrophoresis 3-9 to 3-12isopropanol precipitation 3-10modifications to thermal cycling program 3-7reactions, preparing 3-4 to 3-6template quantity 3-4

Ccapillary electrophoresis, See electrophoresiscentrifuging

384-well plates 6-396-well plates 6-3

chemistryBigDye primer, 384-well format 4-11 to 4-15

cycle sequencing 4-14preparing extension products 4-15preparing reactions 4-12 to 4-13sample clean-up 4-15

BigDye primer, 96-well format 4-3 to 4-9cycle sequencing 4-6 to 4-7preparing extension products 4-8 to 4-9preparing reactions 4-4 to 4-5reaction clean-up 4-8 to 4-9

BigDye terminator, 384-well format 3-13 to 3-21cycle sequencing 3-17extension products, preparing for

Index-2

electrophoresis 3-18 to 3-21preparing reactions 3-14 to 3-16reaction clean-up 3-18 to 3-21

BigDye terminator, 96-well format 3-3 to 3-12 extension products, preparing for

electrophoresis 3-9 to 3-12cycle sequencing 3-7preparing reactions 3-4 to 3-6reaction clean-up 3-9 to 3-12

dRhodamine terminator, 384-well format 5-11 to 5-16cycle sequencing 5-14extension products, preparing for


dRhodamine terminator, 96-well format 5-3 to 5-9cycle sequencing 5-6extension products, preparing for


troubleshooting C-1 to C-2types supported 2-2

conductivity meter, for deionizing formamide A-4customer support

e-mail address B-1help B-1 to B-5Internet address B-5telephone/fax B-2 to B-4

cycle sequencing384-well format

BigDye primer reactions 4-14BigDye terminator reactions 3-17dRhodamine terminator reactions 5-14

96-well formatBigDye primer reactions 4-6BigDye terminator reactions 3-7dRhodamine terminator reactions 5-6

Ddeionized formamide

See formamidedetergent, effect on template quality 2-7Documents on Demand B-5dRhodamine terminator reactions

384-well format 5-11 to 5-16ethanol/sodium acetate precipitation 5-15extension products, preparing for

electrophoresis 5-15reactions, preparing 5-12 to 5-13template quantitiy 5-12volume capacity restrictions 5-12

96-well format 5-3 to 5-9control DNA 5-4cycle sequencing 5-5ethanol/sodium acetate precipitation 5-8extension products, preparing for

electrophoresis 5-7 to 5-9

preparing reactions 5-4 to 5-5template quantity 5-4

dye terminator chemistriesSee BigDye terminator reactions 5-3See dRhodamine terminator reactions 5-3

Eelectrophoresis

advantages of capillary 2-5capillary versus slab gel 2-4optimizing 6-6 to 6-9

e-mail, address for technical support B-1ethanol precipitation

BigDye primer reactions384-well format 4-1596-well format 4-8

BigDye terminators384-well format 3-1996-well format 3-11

ethanol/sodium acetate precipitation384-well format 5-1596-well format 5-8

extension products, preparingBigDye primer

384-well 4-1596-well 4-8 to 4-9

BigDye terminator384-well 3-18 to 3-2196-well 3-9 to 3-12

dRhodamine terminator384-well 5-15 to 5-1696-well 5-7 to 5-9

Fformamide

definition A-1deionizing A-1 to A-5effects of air exposure A-1 to A-2problem with commercial types A-1purchasing Hi-Di formamide A-1storing A-5storing samples in 6-2

Ggel electrophoresis, See electrophoresis

Hhelp

e-mail address B-1Internet address B-5telephone hours B-1telephone/fax B-2 to B-4

humidity, in the laboratory 6-9

Iinjecting samples

Index-3

384-well format 6-396-well format 6-2

injection time 6-4injection voltage 6-4instrument

chemistries supported 2-2factors affecting performance 2-6overview 2-2 to 2-4plates, compatible 2-10run cycle 2-3

Internet addressDocuments on Demand B-5

ion exchange resin, for deionizing formamide A-3isopropanol precipitation


Llaboratory humidity 6-9laboratory temperature 6-9

Mmanuals, part numbers 1-2MicroAmp 384-Well Reaction Plate 2-10MicroAmp Optical 96-Well Reaction Plate 2-10modifications, to thermal cycling protocol 3-7

PPAC, cycle sequencing 3-8part numbers

cycle sequencing kits 2-2formamide deionization reagents A-3Hi-Di formamide A-1POP-6 polymer 2-5user’s manuals 1-2

plate column purification384-well 3-1896-well 3-9

plates384-well 2-1096-well 2-10

polymerSee POP-6 polymer

POP-6 polymerabout 2-4part number 2-5run temperature 6-8run voltage 6-8

primers, modifying cycling parameters based on 3-7protein, effect on template quality 2-7

Rreactions, preparing

BigDye primer, 384-well0.5X reaction 4-131X reaction 4-13

BigDye primer, 96-well

1X reaction 4-52X reaction 4-5

BigDye terminator, 384-wellfor plate column purification 3-15for precipitation 3-16

BigDye terminator, 96-well1X reaction 3-52X for BACs, PACs, YACs 3-62X for bacterial genomic DNA 3-6

dRhodamine terminator, 96-well1X reaction 5-5

resolution, poor, how to resolve 6-5rhodamine dye terminators, See dRhodamine terminator

reactionsRNA, effect on template quality 2-7run temperature 6-8run time

changing for 36-cm array 6-7changing for 50-cm array 6-6

run voltage 6-8

Ssafety 1-3

See also warningssalt, effect on template quality 2-6sample injections


signal, too strong 6-4signal, too weak 6-4spin column, See plate column purification

Ttechnical support B-1 to B-5

e-mail address B-1Internet address B-5telephone/fax B-2 to B-4

temperaturein the laboratory 6-9of the run 6-8

template qualityresidual detergent effect 2-7residual protein effect 2-7residual RNA effect 2-7residual salts effect 2-6

template quantityBigDye primer reactions


BigDye terminator reactions384-well format 3-1496-well format 3-4

dRhodamine terminator reactions384-well format 5-1296-well format 5-4

effect of excess 2-8template, modifying cycling parameters based on 3-7terminator reactions

Index-4

See BigDye terminator reactionsSee dRhodamine terminator reactions

thermal cyclersfor 384-well format 3-17for 96-well format 3-7

thermal cycling, modifications to BigDye terminator 3-7troubleshooting C-1 to C-2

Uuser’s manuals, part numbers 1-2

Vvoltage, injection 6-4voltage, run 6-8volume restrictions, 384-well format

BigDye primer reactions 4-12, 4-15BigDye terminator reactions 3-14dRhodamine terminator reactions 5-12

volumes, samplefor 384-well plate 6-2for 96-well plate 6-2

WWWW address

Applied Biosystems B-5Documents on Demand B-5

YYAC, cycle sequencing 3-8

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