5 replies to this topic

[teerachild]

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 , teerachild@yahoo.com
Thank you.

[Leong Wai Fook]

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.

[teerachild]

Thank you. I'll try.

[Leong Wai Fook]

Good luck

[teerachild]

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?

[moreyroger]

Just use it.  

If it doesnt work, try touchdown from 51-43C.