Chapter 5 Generating Data from RQ Plates – 7500 Fast System
Preparing the Reaction Plate
42 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
FAST
FAST
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:
CHEMICAL HAZARD. TaqMan Universal PCR Master Mix may cause eye and skin irritation.
Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
Samples and endogenous controls are arranged on three plates as shown below. 20 µL of Fast PCR master mix containing
cDNA are added to each well.
The reactions are kept on ice until the plates are loaded on the 7500 Fast system.
Note: To ensure optimal results, run the reaction plate as soon as possible after completing the reaction setup. If you
cannot run a reaction plate within 2 hours after completing the reaction setup, refrigerate or freeze the reaction plate until
you can load and run it on the 7500 Fast instrument.
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
TaqMan Fast Universal PCR Master Mix
(2✕)
10 50 1✕
20✕ TaqM an
®
Gene Expression Assay
Mix
‡
‡ Contains forward and reverse primers and labeled probe.
15 1✕
cDNA sample
945
50 ng (for the 50-µL reaction)
Nuclease-free water —
Total 20 100 —
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
To Next Page
Loading...
Need help?
General
