degenerated primer for nested RT PCR - (Jan/10/2002 )
Dear all, I'm a master degree student. This is my first time to do nested RT-PCR and I made primers for PCR already but I'm not sure that the primers will work coz they are degenerated primers. Well, I asked my advisor and he said it's OK. but I think the problem would occur coz they are high different in Tm for each possible primers. Please email me , firstname.lastname@example.org
It's OK especially for the RT part since the temp for it is low. But for PCR, you have to compromise the Tm for the one of the lowest. Usually choosing a lowest tm for degenerate primers works. For my case, I have Tm as low as 45C and it works. Just try and see.
Thank you. I'll try.
But if the company sent me primer pool in one tube and there's only one annealing temp, 46 c. Should I lower the temp or just use it?
Just use it.
If it doesnt work, try touchdown from 51-43C.