scaling pcr workflows from benchtop to...

Scaling PCR Workflows from

Benchtop to Automation

Broadcast Date: Tuesday, June 29, 2010

Time: 1:00 PM EDT

Sponsored by



Sponsored By



Your Moderator

John SterlingEditor-in-Chief

Genetic Engineering & Biotechnology News

Sponsored By



Lawrence J. Wangh, Ph.D.,Professor of Biology

Laboratory of Molecular Medicine and Global Health

Brandeis University

“Sample Prep for Single Cell Single-Tube LATE-PCR”

Laboratory of Molecular Medicine and Global Health

Department of Biology, Brandeis University, Waltham, MA,

June 29, 2010

Genetic Engineering and Biotechnology News

Webinar

Lawrence J. Wangh, Ph.D.

Pre-Implantation

Genetic

Diagnosis

Early Cancer

Diagnosis

1997 - Small Sample Size

A Challenge to Conventional PCR

Limitations of Real-Time PCR

• Low Sensitivity for Small

Number of Initial DNA Targets

• No Quantitative End-Point

Analysis!

Freeman et al., (1999) Biotechniques 26: 122-125

CT Value

Sample Preparation

in a single tubeImproved SpecificityPrimeSafe

PurAmp&

QuantiLyse

A Whole System ApproachEfficient Amplification

of single-stranded DNA

Dilute’N Go

SequencingVirtual Sequencing

Quantitative End-Point

AnalysisRT –LATE-PCR Multiplexing

Uncoupling ofAnnealing and

Detection

These New Chemistries Result in New Assay Strategies

Lights On/Lights Off Probes

LATE-PCR

see LATE-PCR.org

Therefore measurements are taken

In real-time, as soon as there are

enough molecules

to detect.

Exponential Amplification: 1,2,4,8.16…..

Amplification is Fast, but unreliable

as it slows down.

detection background

real-time

end-point

Linear After the Exponential (LATE)

Amplification is fast and is reliably

It switches from exponential to linear at

shortly after the detectable level is

reached. Reduced scatter at end-point

detection background

real-time

Sample Preparation

in a single tubeImproved SpecificityPrimeSafe QuantiLyse



Dilute’N Go





Detection



LATE-PCR

Pierce, K., Rice, J., Sanchez, J.A., and Wangh, L.J. (2002) “QuantiLyse: Reliable

DNA Amplification from Single Cells”, BioTechniques 32: 1106-1111.

The Problem of Scatter at the

CT Among Single Cells

Pierce et al., (2000). Mol Hum Reprod 6:1155-1164

Optimizing Single-Tube Preparation of Genomic DNA

Chromosome

Packing Density

1:10,000

3 billion base-pairs/CellXY =

In Humans

46

Chromosomes

Per

Somatic Cell

XX =

QuantiLyse: A Simple Reagent and Method for Reliable Preparation of

Genomic DNA in a Single-Tube Reaction

32

34

36

38

40

42

44

0 10 20 30 40 50 60 70 80

CT V

alu

e

Heat Denature

in Water

Freeze/Thaw

in WaterQuantiLyse

34.4

0.34

35.0

0.66

39.4

2.58

Variations in CT Values Among Replicate Reactions

Reflect Variations in Template Accessibility

Single Cell, Single Gene, Single Allele

Analysis via Symmetric PCR

Normal Signals 1278 Signals Aberrant Signals- -

-100

400

900

1400

1900

Flu

oresc

en

ce

Normal/NormalA Normal/1278C

-100

400

900

1400

1900

11 15 19 23 27 31 35 39 43 47 51 55 59

Cycle Number

Flu

oresc

en

ce

1278/1278B

11 15 19 23 27 31 35 39 43 47 51 55 59

Cycle Number

D Normal/1278

Rice et al. (2002), Prenat Diagn 22: 1130-1134

Quality of Genomic DNA Matters!DNA Shearing Increases Scatter

FROZEN/THAWED GENOMIC DNA

Triplex Reaction 2

INTACT GENOMIC DNA: prepared and amplified in the same tube

Triplex Reaction 1

Sample Preparation

in a single tubeImproved SpecificityPrimeSafe QuantiLyse



Dilute’N Go





Detection



LATE-PCR

John Rice, J. Aquiles Sanchez, Kenneth E. Pierce, Arthur H. Reis, Adam Osborne, and

Lawrence J. Wangh (2007) Monoplex/Multiplex Linear-After-The-Exponential (LATE)-PCR

Assays Combined with PrimeSafe® and Dilute-„N‟-Go Sequencing, Nature Protocols 2 #10,

2429-2438.

Yes PrimeSafe™

No PrimeSafe™

Quantitative End-Point LATE-PCR

• Low Scatter Among

Replicates

• High Sensitivity Even for

Low Numbers of Targets

-50

0

50

100

150

200

250

300

350

5 10 15 20 25 30 35 40

Cycle Number

TE

T-F

luorescen

ce (

Rn)

10 g

100 g

1000 g

1/25/05

-50

0

50

100

150

200

250

300

350

5 10 15 20 25 30 35 40

Cycle Number

TE

T-F

luorescen

ce (

Rn)

10 g

100 g

1000 g

1/25/05

500

1000

1500

2000

2500

3000

20 30 40 50 60 70 80

Cycle Number

Fluo

resc

ence

0

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

500

1000

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2500

3000

20 30 40 50 60 70 80

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Fluo

resc

ence

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

0

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1000

1500

2000

2500

3000

20 30 40 50 60 70 80

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0

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

20 30 40 50 60 70 80

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Fluo

resc

ence

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

00

500

1000

1500

2000

2500

3000

20 30 40 50 60 70 80

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Fluo

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1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

500

1000

1500

2000

2500

3000

20 30 40 50 60 70 80

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Fluo

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ence

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

00

500

1000

1500

2000

2500

3000

20 30 40 50 60 70 80

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Fluo

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0

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

20 30 40 50 60 70 80

Cycle Number

Fluo

resc

ence

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

0000

500

1000

1500

2000

2500

3000

20 30 40 50 60 70 80

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Flu

ore

scen

ce

0

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

500

1000

1500

2000

2500

3000

20 30 40 50 60 70 80

Cycle Number

Flu

ore

scen

ce

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

0

500

1000

1500

2000

2500

3000

20 30 40 50 60 70 80

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Flu

ore

scen

ce

0

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

20 30 40 50 60 70 80

Cycle Number

Flu

ore

scen

ce

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

00

500

1000

1500

2000

2500

3000

20 30 40 50 60 70 80

Cycle Number

Flu

ore

scen

ce

0

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

500

1000

1500

2000

2500

3000

20 30 40 50 60 70 80

Cycle Number

Flu

ore

scen

ce

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

00

500

1000

1500

2000

2500

3000

20 30 40 50 60 70 80

Cycle Number

Flu

ore

scen

ce

0

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

20 30 40 50 60 70 80

Cycle Number

Flu

ore

scen

ce

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

10,000 Genomes

1,000 Genomes

100 Genomes

10 Genomes

0000

• Quantitative End-PointAnalysis

Sample Preparation

in a single tubeImproved SpecificityPrimeSafe PurAmp&



Dilute’N Go





Detection



LATE-PCR

Hartshorn C, Anshelevich A, Wangh LJ. (2005) Rapid, single-tube method for

quantitative preparation and analysis of RNA and DNA in samples as small as one

cell. BMC Biotechnol, 5:2.

10,000 kilometers

Separation of the Parts, Rather Than Purification

100,000X100,000X

The Single-tube PurAmp Method

8 – Cell XX 16 – Cell XX 16 – Cell XY Blastocyst XX

Sheardown et al. Cell 91, 99-107 (1997)

Xist Gene Expression and Nuclear Localization

in Developing Mouse Embryos

PurAmp is Accurate and Sensitive

Hartshorn et al. (2005) BMC Biotechnology 5:2

Detection of Xist and Sry in Blastocyst Stage Embryos

Hartshorn et al. (2005) BMC Biotechnology 5:2

Laser Zona Drilling for Single Cell Isolation

Does it cause heat shock?

Hartshorn et al. (2003) Mol Reprod Dev 64:41-51

Quantitative Analysis of Hsp 70 Levels

in Single Blastomeres and Whole

Embryos using the PurAmp Method

Hartshorn et al. (2005) Fertility and Sterility 84, no. 5 1547-1550

Summary of Advantages of Single Tube Sample Preparation

• Separation of Components, Rather than Complete Purification

• Experimentally Convenient and Less Expensive

• Quantitatively More Reliable

optimized for minimum scatter among replicates

• Lower Risk of Laboratory Contamination

• Lower Risk of Sample Contamination

• Gentler on substrates, less shear, less degradation

• Fast and Automatable

Summary of Our Synergistic Core Technologies

• LATE-PCR: abundant, reliable, single-strand production

• Dilute’N’Go Sequencing, more convenient, less costly

• Quantitative End-Point Analysis, cheaper, fewer errors

• PrimeSafe, cleaner results and easier multiplexing

• Low Temperature Sequence-Specific Probes

• Low Temperature Mis-match Tolerant Probes

• Lights-On/Lights-Off Probes – high resolution

analysis and “virtual sequencing” in a closed tube

All of the above chemistries are automatable!

Sponsored By



Gregory L. Shipley, Ph.D.,Assistant Professor

Director, Quantitative Genomics Laboratory

The University of Texas Health Science Center, Houston

Scaling Workflows from Bench Top to Automation

Utilizing Automation for Real-Time qPCR

Gregory L. Shipley, Ph.D.

What do we mean by ‘Automation’?

• Automation refers to using liquid handling robots for

component assembly instead of processing a work flow

manually

• Can be as simple as aspirating and dispensing liquid

from plate A into plate B (or C, D, ... N)

• Can be as complex as automating every step of a

complex workflow utilizing multiple instruments

• Many different kinds of instruments can be integrated

with robot software

A Robotic Workstation - Components

Instruments (l-r)

1-Cytomat 1

2- DTX-880

3- FX- dual arm

96-tip head &

Span-8

4- Cytomat 2

5- ELx405 plate

washer

(not shown)

A Robotic Workstation - Complete

Work Station

inside a

Biosero hepa

filtered hood -

aseptic

environment

siRNA &

compound

screening with

live cells or

biochemical

assays

Why use automation?

• Automation brings a level of accuracy and precision

to an experiment that can not be achieved manually

for large numbers of repetitions

• For small tasks, 1 or 2 96-well plates, it is faster and

can be just as accurate to do the process manually

• However, using automation means that every plate

will be processed the same, every time regardless of

the number of repetitions, complexity or time

required for the task

What is Involved in using Automation?

• Aside from acquiring the instrumentation, learning to

use the software in a sophisticated way is a critical step

(loops, nested loops, IF statements, variables, etc)

• Requires a dedicated person

• Make sure your assay can be scaled down to 96- or

384-well plates (1536)

• Real-Time qPCR lends itself to this format quite nicely

but not true of all assays

What Liquid Handling Robot to Buy??

• Don’t consider just what you need today, think about the

future

• Make sure the features give you all the flexibility you require

1- How many tips (1, 4, 8, 96, 384) or tools (1 tip or 8 tips)

2- The more tips, the faster the job will be done

3- Span-8 capability = maximum flexibility & speed

4- Using 96 or 384 tip heads are fast but not as flexible -

work best with another robot

Pipetting with a Liquid Handling Robot

• By default, robot software set to maximize speed but this

minimizes accuracy

• Slow down aspiration & dispense, volume dependent

• Put in delays, 500 - 1500 ms for aspiration - dispense

• Aspirate (2X), dispense- source (1/2X), dispense- target(s)

(1X), dispense remaining- source

• Pre-wet tips if necessary, depends on solution

• Use appropriate size tips/volume

• Use food color dyes for initial program check

• Use tartrazine (10 mM) to check accuracy/absorbance A427

read - A650 background = 5% - 10% CVs

Working with 96-Well Plates

• Originally started setting up 96-well qPCR plates with a

Biomek 2000 in 1996

• Used a single channel tool, one well at a time, due to

asymmetric layout of the RT reactions

• 45 minutes+ to set up one 96-well plate - over an hour to

run the RT reaction on a thermocycler

• Added PCR master mix with the 8-channel tool

• Almost 2 H for real-time qPCR on the ABI 7700

• State of the art at the time

Biomek2000 with Single Channel Tool

Original 96-Well Plate Layout

Std 1 Std 2 Std 3 Std 4

Std 5 NTC

S #1

S #7

S #1S #1 S #1

S #7 S #7 S #7 S #14 S #14 S #14S #14

S #20 S #20 S #20 S #20

NAC

Sample

Standard

NTC

ASPrimer

4 μl+6 μl

50 μl PCR

Second Generation 96-Well Plate

Std 1 Std 2 Std 3 Std 4 Std 5 NTC

S #1 S #8S #1S #1 S #1 S #8 S #8 S #8 S #15 S #15 S #15S #15

S #21 S #21 S #21 S #21

Data from the 96-Well Plate

A Second Assay: 96-Well Plate

Working with 96- & 384-Well Plates

• 1999 shifted to a Tecan Genesis 100, with Span-8

• Span-8 means the tips can expand/contract in the y-axis

and move independently in the z-axis

• Span-8 allows use of up to 8 tips at once vs 1 previously

• Uses fixed tips, no disposable tip costs

• Use 5% Clorox bleach & H2O washes to clean tips

between samples

• Cut the RT setup time to 17 minutes/plate (384)

• Worth every cent

Tecan Genesis 100 with Span-8

384-Well Plate- Multiple Sample Layouts

NTC + Standard Curve, 1 Assay/Plate NAC for each Sample

15 Samples 30 Samples 45 Samples 61 Samples 71 Samples 93 Samples

5 5 4 4 3 3 2 2 1 1

1 1 1 1

93 93 93 93

NTC NTC

NAC

Sample

Standard

NTC

ASPrimer

2 μl+3 μl

20 μl PCR

Two Assays/Plate- 15, 30 or 45 Samples/Half Plate 1 or 2 Sample Sets

NTC + Standard Curve - 2 Assays, 1 plate NAC for each Sample

15, 30 or 45 Samples, Assay 1, Sample Set 1 15, 30 or 45 Samples, Assay 2, Sample Set 1 or 2

384-Well Plate Data

ABI 7900HT - Human Probe-based CyclinD1 Assay

Whole-Cell Lysates vs Purified RNADMSO vs Staurosporine

• Comparison of making cDNA from whole cell lysates

vs purified RNA

• Treated cells with Staurosporine or DMSO as carrier

• Made cDNA and added to qPCR SYBR master mix

• One 384-well Tox array (Lonza/Bar Harbor Biotech)

per cell culture

• Used 4 cultures for each group

• Total plates is 16

• Loaded plates utilizing a Biomek2000, single

aspirations with quad dispenses, 8-channel tool

Whole-Cell Lysates vs Purified RNADMSO vs Staurosporine

Purified RNA

Cell Lysate

18SrRNA

18SrRNA

19 significant

transcript changes

shared between cell

lysates and purified

RNA that validated

with individual

qPCR assays

Automation

essential for this

experiment

Summary

• Choose the robot platform that fits your workflow,

keeping the future in mind

• Become expert at using the software, dedicated user

• Robots are excellent for repetitive, large scale tasks

• Robots are not good if the work flow changes often

• Using the same robot for plasmid or nucleic acid

preps and setting up qPCR is not a good idea

• Always perform QC on new assays before running

real samples

Sponsored By



David Knorr, Ph.D.,Applications Manager

Automation Solutions Instruments

Agilent Technologies, Inc.

29 June 2010

Agilent - GEN

Scaling PCR

Automation

• Automation rationale, planning and considerations

• PCR workflow

• Different scales of automation

– single liquid handler

– workstation

– integrated system

29 June 2010

Agilent - GEN

55

David Knorr, Ph.D.

Applications Manager

Agilent Technologies

29 June 2010

Agilent - GEN

Agilent Technologies

PCR Workflow Solutions

• Instruments for workflow automation

– plate sealer, centrifuge, labeler, stackers, and more

– liquid handlers

› Bravo

› Vertical Pipetting Station (VPrep)

• BenchCel Workstations

• BioCel fully automated systems

• VWorks software

• Reagents

– PCR polymerases (5 choices)

– PCR / qPCR amplification kits

– purification kits for RNA / DNA / PCR product cleanup

29 June 2010

Agilent - GEN

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29 June 2010

Agilent - GEN

Automation Rationale

• Capacity or throughput improvements

– shrink research and development timelines

• Quality (product and data) improvements

– process standardization / uniformity

– improve consistency

› reduce error

› reduce subjective data analysis

› enable high-density well formats (e.g. 1536 well plates

• Reduce Operating Costs

• Redistribute brain power - scientists are expensive liquid handlers

29 June 2010

Agilent - GEN

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29 June 2010

Agilent - GEN

Plan to Automate

(let the process drive)

• Understand your process;

focus on where you’re going and why

• Scope, Timeline and Cost

– hold onto one and the others will fall into place!

• Review:

– current methods

– up / down stream compatibility and bottlenecks

– flexibility

• A bad process = a bad automated process

29 June 2010

Agilent - GEN

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Scope

TimeCost

29 June 2010

Agilent - GEN

Collaborate with a Good Vendor

• Good components

– ease-of-use, loading, teaching, monitoring, cleaning

– reliable, accessible

– safe

• Good track record

• Good people

– project management

– engineering

– software

– technical support

– service

• Be clear with requirements, communicate often

• Identify a super-user

• Use the system immediately

29 June 2010

Agilent - GEN

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29 June 2010

Agilent - GEN

PCR Workflow

Real-time PCR

Traditional PCR

29 June 2010

Agilent - GEN

Nucleic Acid Purification / Isolation

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NAPI

• Source material from anything: cells, tissues, fruits, bone fragments, etc.

Each has particular challenges

• Once converted into a liquid (or semi-liquid) nucleic acids can be isolated

usually by some form of affinity chemistry

– magnetic beads

› oligo(dT) or other nucleic acid-binding surface

› requires magnet station & plate-handling robot

– silica-based columns (total nucleic acid DNase or RNase)

› usually require spinning, or vacuum (robotics)

– ChargeSwitch® technology

• Dedicated automation available

– rarely perform all steps

– formats may not fit remainder of workflow

29 June 2010

Agilent - GEN

Reaction Setup

Determine process strategy

• One sample - many reactions

• Many samples - ~1 reactions

• Layout considerations (tips, reagents, cooling, primers, master mix, etc.)

• Applications using similar workflows:

– conventional sequencing

– RNAi library transfection

– magnetic bead purifications

• Capping / sealing

– downstream processing

29 June 2010

Agilent - GEN

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Setup

29 June 2010

Agilent - GEN

PCR Setup: can your head do this?

• Single tip mode - templates in column 1

• Row mode - primers + master mix in row 8

• Aliquot master mix - single row of tips

• Aliquot templates - single column

29 June 2010

Agilent - GEN

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Setup

1

2

3

29 June 2010

Agilent - GEN

Agilent PCR Workflow Components

Real-Time PCR

Traditional PCR

29 June 2010

Agilent - GEN

Bravo Handles PCR Workflow

29 June 2010

Agilent - GEN

65

• Small, versatile, lab-friendly footprint

– hood compatible, easy cleaning

• VWorks software

• Gripper robot

• Quick-change pipetting heads

– 96 & 384 disposable & fixed tip

– pin tool (V&P Scientific)

– high accuracy / precision

– 300 nl – 250 µl range

– SBS plates to 1536 wells

– columns / rows

– tube – plate

– tip tracking

• Many accessories

29 June 2010

Agilent - GEN

Robots Enable Integrated Platforms:

Workstations & Systems

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Agilent - GEN

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BenchCel Direct Drive Robot

29 June 2010

Agilent - GEN

BenchCel Workstations

• 1 – ~4 instruments

• Low complexity assays or protocols

• Complex arrangements possible

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29 June 2010

Agilent - GEN

WorkStation for PCR setup

• Bravo

• BenchCel 6R

• BenchCel 4R

• PlateLoc

• Plate Centrifuge

• Multidrop Combi

29 June 2010

Agilent - GEN

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29 June 2010

Agilent - GEN

BioCel Systems: Maximum Throughput,

Hands-Off Automation

29 June 2010

Agilent - GEN

69

• Direct Drive Robot

• VWorks scheduler

– event-driven

– error-handling

– 3rd party drivers

• Environmental control

• Customized protocols

• Limitations:

› space

› budget

› imagination

BioCel 1200

BioCel 1800

29 June 2010

Agilent - GEN

High-Throughput Genotyping BioCel

• Hardware

– dual enclosures

› 3 liquid handlers (magnetic stations and tip washers)

› seal, X-peel, & spin

› 4°C plate storage

(reagents and primers, 189 plates)

› multiple plate stackers

– 10 thermocyclers

– environmental control

• Protocols

– PCR sample preparation

– RT-PCR clean-up

– sequencing-ready amplicons

29 June 2010

Agilent - GEN

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29 June 2010

Agilent - GEN

384,000 PCRs from raw samples? Today?

No problem!

› 5 V11 robots, 1 Staubli robot, 2 translators

› 6 VPreps, 1 Tecan Evo

› 5 Thermo Multidrops, 2 Deerac Equators

› 4°C Liconic tube storage

› BioMicroLab XL9 tube reformatting

› 5 VSpins

› 2 PlateLocs

› 2 VCodes

› 1 computer (VWorks)

29 June 2010

Agilent - GEN

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