Applied Biosystems 7500 Getting Started Guide

Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System
Preparing the Reaction Plate
24 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
STANDARD
STANDARD
STANDARD
Sample Experiment
Primers and probes for the example RQ experiment are obtained from the TaqMan Gene Expression Assays product line
and are provided as a 20✕ Gene Expression Assay Mix. The PCR master mix is prepared as follows:
Samples and endogenous controls are arranged on three plates as shown below. 50 µL of PCR master mix containing
cDNA are added to each well.
The reactions are kept on ice until the plates are loaded on the 7300/7500/7500 Fast system.
Reaction Component
µL/
Sample
µL/ 5 Reactions
§
§ 24 master mixes are prepared, one for each of 23 genes plus the endogenous control. Volume for five reactions (4 replicates plus extra)
to account for pipetting losses.
Final Concentration
TaqMa n
®
Universal PCR Master Mix (2✕) 25.0 125.0 1✕
20✕ Gene Expression Assay Mix
‡
‡ Contains forward and reverse primers and labeled probe.
2.5 12.5 1✕
cDNA sample 5.0 25.0 50 ng (for the 50-µL reaction)
Nuclease-free water 17.5 87.5 —
Total 50.0 250 —
GR2323
Regulus
Liver_96Plate.eps
GR2323
Endogenous
controls (GAPDH)
Liver
samples
GR2324
Regulus
Kidney_96Plate.eps
GR2324
Endogenous
controls (GAPDH)
Kidney
samples
GR2325
Regulus
Bladder_96Plate.eps
GR2325
Endogenous
controls (GAPDH)
Bladder
samples

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)