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Applied Biosystems 7500 Getting Started Guide

Applied Biosystems 7500
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Contents
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide v
Relative Quantification 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 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
About RQ Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Chapter 2 Designing an RQ Experiment 7
Selecting the PCR Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Specifying the Components of an RQ Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Selecting the Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Selecting One- or Two-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Choosing the Probes and Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Chapter 3 Performing Reverse Transcription 17
Guidelines for Preparing RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Converting Total RNA to cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Chapter 4 Generating Data from RQ Plates –
7300 or Standard 7500 System 21
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Preparing the PCR Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Preparing the Reaction Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Creating a Relative Quantification (RQ) Plate Document . . . . . . . . . . . . . . . . . . . . . 25
Specifying Thermal Cycling Conditions and Starting the Run . . . . . . . . . . . . . . . . . 30
Analyzing and Viewing RQ Plate Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Exporting RQ Plate Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

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Applied Biosystems 7500 Specifications

General IconGeneral
Detection MethodFluorescence
Sample Capacity96-well plate
Thermal CyclerYes
Temperature Uniformity±0.5°C
TypeReal-Time PCR System
Thermal Accuracy±0.25°C
Detection Channels5
Weight45 kg
Power Requirements100-240 V, 50/60 Hz
Dynamic Range9 logs
Reaction Volume Range10-100 µL
Temperature Range4°C to 100°C
Thermal Cycler Block96-well
SoftwareSDS Software
DetectionMultiplex (up to 5 colors)

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