RT-PCR Protocol 1.
1. cDNA synthesis
500ug/ml Oligo(dT)12-18 1 ul
10 mM dNTPs 1 ul
Add 0.5-2 ug RNA, and make up to 12 ul with
sterile water
Heat to 70 centigrade for 10 minutes
Chill onto ice directly
Briefly centifuge
Add DEPC water 1 ul
5x first strand buffer
4 ul
0.1 M DTT 2 ul
Mix the vials gently
Incubate at 42 centigrade for 2 minutes
Add Super III ( or other retrotranscriptase) 1 ul
Incubate at 42 centigrade for 50 minutes
70 centigrade for 15 minutes
The synthesized cDNA can be used for following PCR procedure,
or kept at minus 20 cetigrade.
2. PCR
cDNA 1 ul
10x PCR buffer 1 ul
10 uM primer 1 1 ul
10 uM primer 2 1 ul
10 mM dNTP 2 ul
ddH2O
12.5 ul
Taq polymerase 0.5 ul (1U)
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Run the following program:
I. 95 centigrade 2 minutes for 1 cycle
II.
94 centigrade 30 seconds
55 centigrade 45 seconds
72 centigrade 1 minute for 30-36 cycles
III.72 centigrade 7-10 minutes for 1 cycle
IV.Hold on 4 centigrade
The PCR conditions, especially the anealing temperature, are changeable according to
the specific gene to
be amplified and the kit you use.
RT-PCR Protocol 2. RT-PCR Protocol
http://www.sanger.ac.uk/PostGenomics/genetrap/protocols/RTPCR.pdf