Applied biosystems 7300 User manual

Introduction

Designing a

Plus/Minus

Experiment

Setting Up the

Reaction Plate

Performing the

Plus/Minus

Pre-Read Run

Generating

Amplification

Data

Performing the

Plus/Minus

Post-Read Run

STANDARD

Applied Biosystems 7300/7500/7500 Fast

Real-Time PCR System

Plus/Minus Assay

Getting Started Guide

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

NOTICE TO PURCHASER:

Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this

document. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use

of this document.

TRADEMARKS:

Applera, Applied Biosystems, AB (Design), ABI PRISM, BloodPrep, MicroAmp, NucPrep, Primer Express, and VIC are registered trademarks and FAM,

PrepMan, ROX, and TAMRA are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries.

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

SYBR is a registered trademark of Molecular Probes, Inc.

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

Part Number 4378652 Rev. A

09/2006

Introduction

Chapter 1

About the

7300/7500/7500 Fast

System

About Plus/Minus

Assays Using an IPC

About

Plus/Minus Assays

Designing a

Plus/Minus

Experiment

Chapter 2

Using TaqMan®

Probe-Based

Reagent

Configuration

Designing the

Probe and Primers

Setting Up the

Reaction Plate

Chapter 3 Preparing DNA Setting Up

the Reaction Plate

Performing the

Plus/Minus

Pre-ReadRun

Chapter 4 The Pre-Read Run Before You Begin

Creating a

Plus/Minus

Plate Document

Performing the

Pre-Read Run

Generating

Amplification

Data

Chapter 5

Creating a

Plate Document for

Sample Amplification

Performing the

Amplification Run

Chapter 6

Performing the

Plus/Minus

Post-Read Run

Performing the

Post-Read Run

Viewing

Plus/Minus

Results

Exporting

Plus/Minus

Plate Data

STANDARD

STANDARD STANDARD

STANDARD

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide iii

Plus/Minus Experiment Workflow

iv Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide

Contents

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide v

Plus/Minus Experiment Workflow iii

Preface vii

How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

Chapter 1 Introduction 1

About the 7300/7500/7500 Fast System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

About Plus/Minus Assays Using an IPC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

About Plus/Minus Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Chapter 2 Designing a Plus/Minus Experiment 7

Using TaqMan®Probe-Based Reagent Configuration . . . . . . . . . . . . . . . . . . . . . . . . 8

Designing the Probe and Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Chapter 3 Setting Up the Reaction Plate 11

Preparing DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Setting Up the Reaction Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Chapter 4 Performing the Plus/Minus Pre-Read Run 17

The Pre-Read Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Creating a Plus/Minus Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Performing the Pre-Read Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Chapter 5 Generating Amplification Data 27

Creating a Plate Document for Sample Amplification . . . . . . . . . . . . . . . . . . . . . . . . 28

Performing the Amplification Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

vi Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide

Chapter 6 Performing the Plus/Minus Post-Read Run 35

Performing the Post-Read Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Viewing Plus/Minus Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Exporting Plus/Minus Plate Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Appendix A Creating Detectors 45

Appendix B Viewing Amplification Data 47

Specifying Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Analyzing the Plus/Minus Amplification Data (AQ Plate) . . . . . . . . . . . . . . . . . . . . . 48

Viewing the Amplification Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Appendix C Example Plus/Minus Experiment 55

References 61

Index 63

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide vii

Preface

How to Use This Guide

Purpose of This

Guide

This manual is written for principal investigators and laboratory staff who run

plus/minus assays using the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR

System (7300/7500/7500 Fast system).

Assumptions This guide assumes that you have:

• Familiarity with Microsoft®Windows®XP operating system.

• Knowledge of general techniques for handling DNA samples and preparing them

for PCR.

• A general understanding of hard drives and data storage, file transfers, and copying

and pasting.

• Networking experience if you want to integrate the 7300/7500/7500 Fast system

into your existing laboratory data flow system.

Text Conventions This guide uses the following conventions:

•Bold indicates user action. For example:

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

•Italic text indicates new or important words and is also used for emphasis. For

example:

Before analyzing, always prepare fresh matrix.

• 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

The following 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.

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.

Preface

How to Use This Guide

viii Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide

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Refer to the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System

Installation and Maintenance Getting Started Guide (PN 4378657) and the

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Site Preparation

Guide (PN 4378654) for important safety information.

Preface

How to Obtain More Information

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide ix

How to Obtain More Information

Documentation

For more information about using the 7300/7500/7500 Fast system, refer to the

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Online Help or the

documents shown below.

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