Genomics Lab/Biology Dept
Standard Experimental Protocol
Title: Setting up TaqMan RT-PCR
Page 1 of 2
SEP No: 009
Date: 13.3.06
Date of Review: 23.02.07
Author: Celina Whalley
Reviewer: Naveed Aziz Version: 2.1
The purpose of a SEP is to enable personnel undertake a defined experimental
procedure
1. Purpose:
To set up a TaqMan plate in preparation for RT-PCR.
2. Scope:
To measure the amount of DNA template generated, over time, to assess specific
gene expression.
3. Safety:
MSDS forms are located in K241 above the ABI7000 machine.
4. Materials
4.1
4.2
4.3
Raw materials: 96 well plates and covers. Applied Biosytems (ABI) part code
4314320
Equipment required: ABI 7000 system
Reagents: TaqMan. ABI part code 4304437
5. Reaction set-up:
a) Reverse Transcription
Total RNA (1 g)
Random hexamer / Oligo dT primers (10 mM)
dNTP mix (10 mM)
Total
X l
1 l
1 l
to 12 l
Incubate the samples at 65°C for 5 min and then on ice for at least 1 min then add;
10x Reverse Transcription Buffer
DDT (0.1 M)
RNase Out (40 u/l)
4 l
2 l
1 l
Mix and incubate @ 42°C for 2 min and add;
Reverse Transcriptase (200U/l)
Total
D:\533561979.doc
Page 1 of 2
1 l
20 l
Genomics Lab/Biology Dept
Standard Experimental Protocol
Title: Setting up TaqMan RT-PCR
Page 2 of 2
SEP No: 005
Date: 13.3.06
Date of Review: 23.02.07
Author: Celina Whalley
Reviewer: Naveed Aziz Version: 2.1
Mix gently and incubate the tubes at 42°C for 1-2 hr
Heat inactivate the enzyme at 70°C for 15 min, and then chill on ice.
Store the 1st strand cDNA at -20°C until use for real-time PCR.
a. Preparing TaqMan PCR Master Mix
Taqman Master Mix (2x)
cDNA (5-50 ng)
Primer pair mix (10 M each primer)
TaqMan probe (2.5 M)
Nuclease free water
12.5 l
5 l (dilution of the above cDNA sample)
2 l
2.5 l
3 l
25l total per reaction
Set up the ABI7000 software as in SOP006 (ABI7000 operation).
After PCR is finished, remove the plate from the machine and dispose of the plate in the yellow
waste bin provided.
6. Training
All users are signed off after completing the lab safety check followed by the training
schedule three times. A list of super-users is located in a folder above the ABI 7000
machine.
D:\533561979.doc
Page 2 of 2