Applied Biosystems 7300 Getting Started Guide

Appendix B Example RQ Experiment
82 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
Example RQ Experiment Procedure
1. Design the experiment, as explained in Chapter 2
on page 7.
a. Designate the targets, endogenous control,
and replicates.
b. Order the reagents for TaqMan
®
probe-
based chemistry.
c. Order the appropriate TaqMan
®
Gene
Expression Assays products, which provide
predesigned primers and probes for the 23
genes.
2. Isolate total RNA from liver, kidney, and bladder
tissue, as explained in Chapter 3 on page 18.
3. Generate cDNA from total RNA using the High
Capacity cDNA Archive Kit.
a. Prepare the reverse transcription (RT)
master mix as indicated in the table to the
right.
Additional guidelines are provided in the
High Capacity cDNA Archive Kit Protocol.
CHEMICAL HAZARD.
10 × RT Buffer may cause eye, skin, and respiratory
tract irritation. Read the MSDS, and follow the
handling instructions. Wear appropriate protective
eyewear, clothing, and gloves.
RT Master Mix - Standard Plate
Component µL/Reaction
µL/21
reactions
‡
‡ Each RT reaction is 100 µL (see step 3b). If you need 5 µL cDNA
for each of 104 PCR reactions per tissue (see step 4), you need 6
RT reactions per tissue. Extra volume (enough for one additional
RT reaction per tissue) is included to account for pipetting losses,
as well as extra cDNA for archiving.
10✕ Reverse
Transcription Buffer
10 210
25✕ dNTPs 4 84
10✕ random primers 10 210
MultiScribe
™
Reverse
Transcriptase, 50 U/µL
5 105
Nuclease-free water 21 441
Total 50 1050

Applied Biosystems 7300 Specifications

General

Brand	Applied Biosystems
Model	7300
Category	Laboratory Equipment
Language	English