5. Use a serial dilution of the reverse transcription reaction: 2ul of undiluted cDNA, 2μl of 10 x diluted cDNA and 2ul of 100 x diluted cDNA in the PCR reaction. Set up the PCR as follows:
35.5μl pure water
5μl 10 x PCR buffer (Boehringer Mannheim)
5μl 500uM dNTPs (some enzymes require a higher concentration of dNTPs (2mM), for example Pfu polymerase (though I'd recommend against the use of this enzyme)
1μl Adapter primer (25uM)
1μl Gene specific primer (25uM)
2μl cDNA template (various dilutions)
6. Mix and overlay with mineral oil.
7. Perform the following PCR cycles:
a. 94℃ 3 minutes
b. 72℃ 3 minutes (at this point add 0.5ul of Taq polymerase)
c. 94℃ 30 seconds
d. 56℃* 1 minute
e. 72℃ 2 minutes (longer if expected fragment size is greater than 2kb)
f. Cycle steps c. - e. 35 times
g. 72℃ 10 minutes.
Varies according to the melting temperature of your gene specific primer. The annealing temperature in the PCR may require some adjustment for optimal yield of PCR product.
8. Load all or part of the reaction on an agarose gel. Don't be too surprised if you get a faint background smear as well as your prominent band.