Thermo Fisher Scientific Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Owner's manual

Applied Biosystems

7900HT Fast Real-Time PCR System

and SDS Enterprise Database

User Guide

DRAFT

November 19, 2007 11:09 am, 4351684A_Title.fm

Applied Biosystems

7900HT Fast Real-Time PCR System

and SDS Enterprise Database

User Guide

DRAFT

November 19, 2007 11:09 am, 4351684A_Title.fm

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

The Applied Biosystems 7900HT Fast Real-Time PCR System is a real-time thermal cycler covered by US patents and corresponding claims in their non-US coun-

terparts, owned by Applied Biosystems. No right is conveyed expressly, by implication or by estoppel under any other patent claim, such as claims to apparatus,

reagents, kits, or methods such as 5’ nuclease methods. Further information on purchasing licenses may be obtained by contacting the Director of Licensing,

Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

TRADEMARKS:

ABI PRISM, Applied Biosystems, MicroAmp, Primer Express, and VIC are registered trademarks and AB (Design), ABI PRISM, Applera, FAM, JOE, NED, ROX,

TAMRA, a n d TET a re tra d e m arks o f Applied Biosystems or its subsidiaries in the US and/or certain other countries.

AmpErase, AmpliTaq Gold, GeneAmp, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.

SYBR is a registered trademark of Molecular Probes, Inc.

Zymark is a registered trademark of Zymark Corporation.

Windows and Windows NT are registered trademarks of Microsoft Corporation.

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

Part Number 4351684 Rev. C

06/2010

DRAFT

November 19, 2007 10:42 am, 4351684ATOC.fm

Contents

Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide iii

Preface

How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

How to Obtain Services and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

Safety and EMC Compliance Information

Safety Conventions Used in This Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

Symbols on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

Safety Labels on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi

General Instrument Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii

Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii

Chemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx

Physical Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Biological Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Laser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Bar Code Scanner Laser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii

Workstation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiii

Safety and Electromagnetic Compatibility (EMC) Standards . . . . . . . . . . . . . . . . . . . . xxiii

Chapter 1 Product Overview

System Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Section 1.1 Getting to Know the Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6

Bar Code Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7

Zymark Twister Microplate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8

Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9

Instrument Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10

Section 1.2 Getting to Know the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-13

Software Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-14

SDS Software Related Files and Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15

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iv Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide

Managing Sequence Detection System Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-17

Section 1.3 SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-21

About the SDS Enterprise Database Feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-22

About the SDS Enterprise Database Software Suite . . . . . . . . . . . . . . . . . . . . . . . . . . 1-25

Database Modules for Large-Scale Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-25

Database Management Utilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-26

Database Design and Information Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-27

Supporting API Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-28

Chapter 2 Getting Started

Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Powering On the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4

Using the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

Basic Software Skills Tutorial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10

Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-11

Lesson 1: Using Plate Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12

Lesson 2: Viewing and Resizing Panes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-21

Lesson 3: Using the Plate Grid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-23

Lesson 4: Using the Hand-Held Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . 2-27

Lesson 5: Using Contextual Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-28

Lesson 6: Using Keyboard Shortcuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-28

Using SDS Plate Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29

Chapter 3 Preparing a Run

Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Quick Review: Powering On the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7

Step 1 – Creating a Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8

Step 2 – Applying Detectors and Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

Step 3 – Configuring the Plate Document with Tasks . . . . . . . . . . . . . . . . . . . . . . . . . 3-14

Step 4 – Setting the Passive Reference and Omitting Wells . . . . . . . . . . . . . . . . . . . . 3-16

Step 5 – Programming the Plate Document Method . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17

Step 6 – Saving the Plate Document as a Template . . . . . . . . . . . . . . . . . . . . . . . . . . 3-22

Step 7 – Creating a Plate Document from the Template . . . . . . . . . . . . . . . . . . . . . . . 3-24

Step 8 – Applying Sample and Plate Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-25

Step 9 – Running the Plate on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . 3-26

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Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide v

Chapter 4 Operating the Instrument

Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Section 4.1 Consumable Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5

Preventing Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Preparing Optical Plates for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Preparing TaqMan Low Density Arrays for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10

About the Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10

About the Centrifuge System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12

About the Sealer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Loading the TaqMan Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Centrifuging the TaqMan Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16

Sealing the TaqMan Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-19

Section 4.2 Running an Individual Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23

Saving the Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-24

Running a Single Plate (Using the SDS Software) . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-25

After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-29

Section 4.3 Automated Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-31

Operating the Software with an SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . 4-32

Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . . . . 4-34

Adding a Plate Document to the Plate Queue (Using the SDS Software) . . . . . . 4-35

Creating Plate Documents Using the Template Batch Utility . . . . . . . . . . . . . . . . 4-36

Starting and Configuring the Automation Controller Software for Operation . . . . 4-39

Running Plates Using the Automation Controller Software . . . . . . . . . . . . . . . . . . . . 4-41

Loading Plates onto the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-41

Running the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-43

After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-44

Chapter 5 Analyzing End-Point Data

End-Point Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2

Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3

Section 5.1 Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5

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

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9

Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10

Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11

Analyzing an Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12

Calling and Scrutinizing Allelic Discrimination Data . . . . . . . . . . . . . . . . . . . . . . . . . . 5-13

After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-18

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vi Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide

Chapter 6 Analyzing Real-Time Data

Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2

Real-Time Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3

Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5

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

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8

Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9

Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10

Analyzing the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11

Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-13

After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14

Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-16

Algorithmic Manipulation of Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-17

Automatic Outlier Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-19

Essential Experimental Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20

Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21

Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22

Options for Analyzing Relative Quantification Data . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23

Creating the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23

Analyzing the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26

About the Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26

Specifying the Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-29

Analyzing the Study Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-30

Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-31

After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-35

Section 6.3 Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-39

Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-40

Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41

Analyzing the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41

Determining Tm Values for the Analyzed Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-42

After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-44

Section 6.4 Procedure Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-45

Setting the Baseline and Threshold Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-46

Eliminating Outlying Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-49

After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52

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Chapter 7 Maintaining the Instrument

Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2

Recommended Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3

Section 7.1 Maintaining the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-5

Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6

Changing the Plate Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12

Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-14

Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16

Preparing the Background Plate or TaqMan Low Density Array . . . . . . . . . . . . . 7-17

Creating a Plate Document for the Background Run . . . . . . . . . . . . . . . . . . . . . . 7-17

Running the Prepared Background Plate or TaqMan Low Density Array . . . . . . 7-18

Analyzing the Background Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-19

Performing a Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20

Preparing the Pure Dye Plates or Cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-22

Preparing a Plate Document for a Pure Dye Plate or Card . . . . . . . . . . . . . . . . . 7-22

Running the Prepared Pure Dye Plate or Card . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-24

Analyzing the Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-25

Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27

Verifying Instrument Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-30

Section 7.2 Maintaining the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-35

Automation Accessory Components and Stack Positions . . . . . . . . . . . . . . . . . . . . . 7-36

Adjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . . . . . 7-37

Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-41

Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-49

Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-52

Section 7.3 Maintaining the Computer and Software . . . . . . . . . . . . . . . . . . . . . . . 7-53

General Computer Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-54

Maintaining the SDS software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-55

Chapter 8 Troubleshooting

Troubleshooting Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2

Low Precision or Irreproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-5

Background Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-9

Pure Dye Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-11

Real-Time Runs (Quantitative PCR and Dissociation Curves) . . . . . . . . . . . . . . . . . . 8-12

End-Point Runs (Allelic Discrimination) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-13

Software and 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-14

Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader . . . . . . . . 8-17

TaqMan Low Density Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-18

SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-20

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viii Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide

Appendix A Software Reference

Importing Plate Document Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2

Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-4

1. File Version . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5

Plate Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5

2. Plate Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5

3. Plate ID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5

Detector Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-6

4. Number of Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-6

5. Detectors List Header . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-6

6. Detectors List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7

Marker Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-8

7. Number of Markers (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . . . . . . . A-8

8. Markers List Header (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . . . . . . A-8

9. Markers List (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-9

Well-Detector Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-10

10. Well-Detector List Header . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-10

11. Well-Detector Definition List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-10

Well-Marker Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-12

12. Well-Marker List Header (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . A-12

13. Well-Marker Definition List (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . A-12

Example Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-14

Exporting Graphics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16

Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16

Operating the SDS Software from a Command Line . . . . . . . . . . . . . . . . . . . . . . . . . . A-18

Using the Search Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-20

Connecting SDS Software to the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-22

Appendix B Designing TaqMan Reagent-Based Assays

Assay Development Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2

Design Tips for Allelic Discrimination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-5

Design Tips for Quantitative PCR Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-6

Appendix C Kits, Reagents, and Consumables

Interchangeable Sample Block Modules and Accessories . . . . . . . . . . . . . . . . . . . . . . C-2

Consumables and Disposables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-3

Instrument Maintenance and Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-5

TaqMan Pre-Developed Assays and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6

Custom Oligonucleotide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6

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Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide ix

Appendix D Theory of Operation

Fluorescent-Based Chemistries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

Real-Time Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-4

Comparative CT Method of Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . D-8

Appendix E Limited Warranty Statement

Bibliography

Glossary

Index

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x Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide

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Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide xi

Preface

How to Use This Guide

Purpose of This

Guide

The Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise

Database User Guide describes how to prepare, maintain, and troubleshoot the

Applied Biosystems 7900HT Fast Real-Time PCR System instrument, Zymark®

Twister Microplate Handler, and SDS Enterprise Database.

Audience This guide is written for technicians, scientists, and researchers of all skill levels who

will use and maintain 7900HT instruments with or without the SDS Enterprise

Database.

Assumptions This guide assumes that a Applied Biosystems technical representative has installed

your 7900HT instrument, Plate Handler, and SDS Enterprise Database and that you

are familiar with:

•Basic Microsoft

® Windows® operations such as using the mouse, choosing

commands, working with windows, and using the hierarchical file system

• Electronic storage devices (computer hard drives) and data files

• Assay preparation and basic laboratory techniques

Text Conventions This guide uses the following conventions:

•Bold indicates user action. For example:

Enter 0, then press Enter for each of the remaining fields.

•Italics indicates new or important words and is used for emphasis. For example:

Before performing a pure dye calibration, always perform a background run.

• A right arrow bracket (>) separates successive commands you select from a

drop-down or shortcut menu. For example:

Select File > Open > Spot Set.

User Attention

Words

Two user attention words appear in Applied Biosystems user documentation. Each

word implies a particular level of observation or action as described below:

Note: Provides information that may be of interest or help but is not critical to the

use of the product.

IMPORTANT! Provides information that is necessary for proper instrument

operation, accurate chemistry kit use, or safe use of a chemical.

Safety Alert

Words

Safety alert words also appear in user documentation. For more information,

see “Safety Alert Words” on page xiv.

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Preface

xii Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide

How to Obtain More Information

See the following related documents for more information on the topics in this guide:

•Applied Biosystems 7900HT Fast Real-Time PCR System Quick Starts –

Provide brief, procedures for performing application-specific tasks on the

7900HT instrument.

•Sequence Detections Systems Software Online Help – Describes the Sequence

Detection Systems (SDS) Software and provides procedures for common tasks.

•SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real-Time

PCR System Administrators Guide – Provides information for database

administrators who will be maintaining the SDS Enterprise Database, and

provides information for systems integrators who will be working with the SDS

Enterprise Database API.

Note: For additional documentation, see “How to Obtain Services and Support” on

page xii.

Send Us Your

Comments

Applied Biosystems welcomes your comments and suggestions for improving its

user documents. You can e-mail your comments to:

[email protected]

How to Obtain Services and Support

To contact Applied Biosystems Technical Support from North America by telephone,

call 1.800.899.5858.

For the latest services and support information for all locations, go to

http://www.appliedbiosystems.com, then click the link for Services and Support.

At the Services and Support page, you can:

• Search through frequently asked questions (FAQs)

• Submit a question directly to Technical Support

• Order Applied Biosystems user documents, MSDSs, certificates of analysis,

and other related documents

• Download PDF documents

• Obtain information about customer training

• Download software updates and patches

In addition, the Services and Support page provides access to worldwide telephone

and fax numbers to contact Applied Biosystems Technical Support and Sales

facilities.

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Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide xiii

Safety and EMC Compliance Information

This section includes the following topics:

Safety Conventions Used in This Document. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

Symbols on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xv

Safety Labels on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi

General Instrument Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii

Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii

Chemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xx

Physical Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Biological Hazard Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Laser Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Bar Code Scanner Laser Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii

Workstation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiii

Safety and Electromagnetic Compatibility (EMC) Standards . . . . . . . . . . . . . . . xxiii

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xiv Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide

Safety Conventions Used in This Document

Safety Alert

Words

Four safety alert words appear in Applied Biosystems user documentation at points

in the document where you need to be aware of relevant hazards. Each alert

word–IMPORTANT, CAUTION, WARNING, DANGER–implies a particular

level of observation or action, as defined below:

Definitions

IMPORTANT! – Indicates information that is necessary for proper instrument

operation, accurate chemistry kit use, or safe use of a chemical.

– Indicates a potentially hazardous situation that, if not avoided,

may result in minor or moderate injury. It may also be used to alert against unsafe

practices.

– Indicates a potentially hazardous situation that, if not avoided,

could result in death or serious injury.

– Indicates an imminently hazardous situation that, if not avoided,

will result in death or serious injury. This signal word is to be limited to the most

extreme situations.

Except for IMPORTANTs, each safety alert word in an Applied Biosystems

document appears with an open triangle figure that contains a hazard symbol. These

hazard symbols are identical to the hazard icons that are affixed to Applied

Biosystems instruments (see “Safety Symbols” on page xv).

Examples

The following examples show the use of safety alert words:

IMPORTANT! You must create a separate a Sample Entry Spreadsheet for each

96-well microtiter plate.

The lamp is extremely hot. Do not touch the lamp until it has

cooled to room temperature.

CHEMICAL HAZARD. Formamide. Exposure causes eye,

skin, and respiratory tract irritation. It is a possible developmental and birth defect

hazard. Read the MSDS, and follow the handling instructions. Wear appropriate

protective eyewear, clothing, and gloves.

ELECTRICAL HAZARD. Failure to ground the instrument

properly can lead to an electrical shock. Ground the instrument according to the

provided instructions.

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Symbols on Instruments

Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide xv

Symbols on Instruments

Electrical

Symbols on

Instruments

The following table describes the electrical symbols that may be displayed on

Applied Biosystems instruments.

Safety Symbols

The following table describes the safety

s

ymbols that may be displayed on

Applied Biosystems instruments. Each symbol may appear by itself or in combination

with text that explains the relevant hazard (see “Safety Labels on Instruments” on

page xvi). These safety symbols may also appear next to DANGERS, WARNINGS,

and CAUTIONS that occur in the text of this and other product-support documents.

Symbol Description Symbol Description

Indicates the On position of the

main power switch.

Indicates a protective

grounding terminal that must

be connected to earth ground

before any other electrical

connections are made to the

instrument.

Indicates the Off position of the

main power switch.

Indicates the On/Off position of

a push-push main power

switch.

Indicates a terminal that can

receive or supply alternating

current or voltage.

Indicates a terminal that may

be connected to the signal

ground reference of another

instrument. This is not a

protected ground terminal.

Indicates a terminal that can

receive or supply alternating or

direct current or voltage.

Symbol Description Symbol Description

Indicates that you should

consult the manual for further

information and to proceed

with appropriate caution.

Indicates the presence of a

laser inside the instrument and

to proceed with appropriate

caution.

Indicates the presence of an

electrical shock hazard and to

proceed with appropriate

caution.

Indicates the presence of

moving parts and to proceed

with appropriate caution.

Indicates the presence of a hot

surface or other

high-temperature hazard and

to proceed with appropriate

caution.

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xvi Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide

Safety Labels on Instruments

The following CAUTION, WARNING, and DANGER statements may be displayed

on Applied Biosystems instruments in combination with the safety symbols

described in the preceding section.

English Francais

CAUTION

Hazardous chemicals. Read the Material Safety

Data Sheets (MSDSs) before handling.

ATTENTION

Produits chimiques dangeureux. Lire les fiches

techniques de sûreté de matériels avant la manipulation des

produits.

CAUTION

Hazardous waste. Read the waste profile (if any)

in the site preparation guide for this instrument before

handling or disposal.

ATTENTION

Déchets dangereux. Lire les renseignements

sur les déchets avant de les manipuler ou de les éliminer.

CAUTION

Hazardous waste. Refer to MSDS(s) and local

regulations for handling and disposal.

ATTENTION

Déchets dangereux. Lire les fiches techniques

de sûreté de matériels et la régulation locale associées à la

manipulation et l'élimination des déchets.

WARNING

Hot lamp.

AVERTISSEMENT

Lampe brûlante.

WARNING

Hot. Replace lamp with an Applied Biosystems

lamp.

AVERTISSEMENT

Composants brûlants. Remplacer la

lampe par une lampe Applied Biosystems.

CAUTION

Hot surface.

ATTENTION

Surface brûlante.

DANGER

High voltage.

DANGER

Haute tension.

WARNING

To reduce the chance of electrical shock, do not

remove covers that require tool access. No user-serviceable

parts are inside. Refer servicing to Applied Biosystems

qualified service personnel.

AVERTISSEMENT

Pour éviter les risques d'électrocution, ne

pas retirer les capots dont l'ouverture nécessite l'utilisation

d'outils. L’instrument ne contient aucune pièce réparable par

l’utilisateur. Toute intervention doit être effectuée par le

personnel de service qualifié de Applied Biosystems.

DANGER

Class 3B laser radiation present when open and

interlock defeated. Avoid direct exposure to laser beam.

DANGER

Class 3B rayonnement laser en cas d’ouverture et

d’une neutralisation des dispositifs de sécurité. Eviter toute

exposition directe avec le faisceau.

DANGER

Class 3B laser radiation when open. Avoid direct

exposure to laser beam.

DANGER

Class 3B rayonnement laser en cas d’ouverture.

Eviter toute exposition directe avec le faisceau.

DANGER

Class 3B laser radiation present when open and

interlock defeated. Do not stare directly into the beam

DANGER

de Class 3B rayonnement laser en cas d'ouverture

et d'une neutralisation des dispositifs de securite. Eviter

toute exposition directe avec le faisceau.

DANGER

Class 3B laser radiation present when open. Do

not stare directly into the beam.

DANGER

de Class 3B rayonnement laser en cas

d'ouverture. Eviter toute exposition directe avec le faisceau.

DANGER

Class 3B LED when open and interlock defeated.

Do not stare directly into the beam.

DANGER

de Class 3B LED en cas d'ouverture et d'une

neutralisation des dispositifs de securite. Eviter toute

exposition directe avec le faisceau.

DANGER

Class 3B LED when open. Do not stare directly

into the beam.

DANGER

de Class 3B LED en cas d'ouverture. Eviter toute

exposition directe avec le faisceau.

CAUTION

Moving parts.

ATTENTION

Parties mobiles.

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General Instrument Safety

Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide xvii

General Instrument Safety

PHYSICAL INJURY HAZARD. Use this product only as

specified in this document. Using this instrument in a manner not specified by

Applied Biosystems may result in personal injury or damage to the instrument.

Moving and

Lifting the

Instrument

PHYSICAL INJURY HAZARD. The instrument is to be moved

and positioned only by the personnel or vendor specified in the applicable site

preparation guide. If you decide to lift or move the instrument after it has been

installed, do not attempt to lift or move the instrument without the assistance of

others, the use of appropriate moving equipment, and proper lifting techniques.

Improper lifting can cause painful and permanent back injury. Depending on the

weight, moving or lifting an instrument may require two or more persons.

Moving and

Lifting Stand-

Alone Computers

and Monitors

Do not attempt to lift or move the computer or the monitor

without the assistance of others. Depending on the weight of the computer and/or the

monitor, moving them may require two or more people.

Things to consider before lifting the computer and/or the monitor:

• Make sure that you have a secure, comfortable grip on the computer or the

monitor when lifting.

• Make sure that the path from where the object is to where it is being moved is

clear of obstructions.

• Do not lift an object and twist your torso at the same time.

• Keep your spine in a good neutral position while lifting with your legs.

• Participants should coordinate lift and move intentions with each other before

lifting and carrying.

• Instead of lifting the object from the packing box, carefully tilt the box on its

side and hold it stationary while someone slides the contents out of the box.

Operating the

Instrument

Ensure that anyone who operates the instrument has:

• Received instructions in both general safety practices for laboratories and

specific safety practices for the instrument.

• Read and understood all applicable Material Safety Data Sheets (MSDSs). See

“About MSDSs” on page xviii.

PHYSICAL INJURY HAZARD. Use this instrument as

specified by Applied Biosystems. Using this instrument in a manner not specified by

Applied Biosystems may result in personal injury or damage to the instrument.

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xviii Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide

Chemical Safety

Chemical Hazard

Warning

CHEMICAL HAZARD.

Before handling any chemicals, refer to

the Material Safety Data Sheet (MSDS) provided by the manufacturer, and observe all

relevant precautions.

CHEMICAL HAZARD.

All chemicals in the instrument, including

liquid in the lines, are potentially hazardous. Always determine what chemicals have been

used in the instrument before changing reagents or instrument components. Wear

appropriate eyewear, protective clothing, and gloves when working on the instrument.

CHEMICAL HAZARD.

Four-liter reagent and waste bottles can

crack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety

container with the cover fastened and the handles locked in the upright position. Wear

appropriate eyewear, clothing, and gloves when handling reagent and waste bottles.

CHEMICAL STORAGE HAZARD

. Never collect or store waste

in a glass container because of the risk of breaking or shattering. Reagent and waste

bottles can crack and leak. Each waste bottle should be secured in a low-density

polyethylene safety container with the cover fastened and the handles locked in the

upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent

and waste bottles.

About MSDSs Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with

shipments of hazardous chemicals to new customers. They also provide MSDSs with

the first shipment of a hazardous chemical to a customer after an MSDS has been

updated. MSDSs provide the safety information you need to store, handle, transport,

and dispose of the chemicals safely.

Each time you receive a new MSDS packaged with a hazardous chemical, be sure to

replace the appropriate MSDS in your files.

Obtaining

MSDSs

You can obtain from Applied Biosystems the MSDS for any chemical supplied by

Applied Biosystems. This service is free and available 24 hours a day.

To obtain MSDSs:

1. Go to www.appliedbiosystems.com, click Support, then click MSDS Search.

2. In the Keyword Search field, enter the chemical name, product name, MSDS

part number, or other information that appears in the MSDS of interest. Select

the language of your choice, then click Search.

3. Find the document of interest, right-click the document title, then select any of

the following:

•Open – To view the document

•Print Target – To print the document

•Save Target As – To download a PDF version of the document to a

destination that you choose

Note: For the MSDSs of chemicals not distributed by Applied Biosystems, contact

the chemical manufacturer.

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Thermo Fisher Scientific Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Owner's manual

Thermo Fisher Scientific Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Owner's manual

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