RT-PCR for various sample with Tm from 45-60 degree - (Jun/09/2008 )
Let say i have 10 samples with Tm ranging from 45 to 60°C and i am running reverse transcription RT-PCR. If i set the Tm to 60°C and run all 10 samples, how will it affects the results? Or must i have run individually for each sample?
Wait... let me get the question right. You have total RNA or mRNA, and you want to do RT-PCR. And for this purpose you have PCR primers with Tm ranging from 45-60. All right, first of all you have to run a gradient PCR with gDNA or cDNA to optimize your primers, I mean to find the annealing temperature. Because the Tm your primer design software gives you is calculated based on a simple definition:
Tm = 2(A + T) + 3(G + C)
But this is not necessarily the annealing temperature optimum for the product you aim for. If you choose too low a Ta, you'll get non-specific amplification. Too high a Ta and you'll get too little product. As I said, run a gradient for each primer pair, starting from 5°C below your Tm, and see what happens. But if the Tm s are so different you probably have to amplifiy them separately.