Applied Biosystems 7500 Getting Started Guide

Chapter 2 Designing an RQ Experiment
Selecting One- or Two-Step RT-PCR
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide 13
Notes
2
Two-Step RT-PCR One-Step RT-PCR
RT Master Mix
Sample RNA
Fast PCR Master Mix
Archive
Results - Fast
<
40 minutes
GR2332
RT Incubation
PCR
amplification
and detection
Standard PCR Master Mix OR
Results - Standard
1 hour, 50 minutes
PCR
amplification
and detection
Aliquot cDNA
RT
Master Mix
Sample RNA
Standard PCR
Master Mix
Results
2.5 hours
GR2333
RT incubation and
PCR amplification
Recommended Kits for Two-Step RT-PCR
Chemistry Step Reagent Part Number
TaqMan reagents or kits RT High Capacity cDNA Archive Kit 4322171
PCR TaqMan Universal PCR Master Mix
TaqMan Fast Universal PCR Master Mix
4304437
4352042
SYBR Green I reagents or kits RT High Capacity cDNA Archive Kit 4322171
PCR SYBR Green Master Mix 4309155
RT and PCR SYBR Green RT-PCR Reagents 4310179
Sample Experiment
Premade probes and primers for all the genes of interest are available from the TaqMan
®
Gene Expression Assays product
line, which uses TaqMan reagent-based chemistry. Two-step RT-PCR is performed using the reagents recommended for
TaqMan reagent- or kit-based chemistry in the table above.

Applied Biosystems 7500 Specifications

General

Detection Method	Fluorescence
Sample Capacity	96-well plate
Thermal Cycler	Yes
Temperature Uniformity	±0.5°C
Type	Real-Time PCR System
Thermal Accuracy	±0.25°C
Detection Channels	5
Weight	45 kg
Power Requirements	100-240 V, 50/60 Hz
Dynamic Range	9 logs
Reaction Volume Range	10-100 µL
Temperature Range	4°C to 100°C
Thermal Cycler Block	96-well
Software	SDS Software
Detection	Multiplex (up to 5 colors)