Protocol Online logo
Top : Forum Archives: : Molecular Biology

Primers - pcr problem (Feb/08/2002 )

Dear all, I have a problem with my primers. My PCR reaction worked well but I large amount of nonspecific product. I tried to increase the annealing temp to 58 C but it didn't work. I have to do sequencing reaction but I'm afraid that I'll get signals from nonspecific sequence. Should I get a new set of primers or do one sequencing reaction using only forward primer, annealing temp = 64 C and forget about the reverse primer coz the annealing temp is only 60 C ?  Annealing temp of reverse primer = 60 C, forward = 64 C.
Thank you.

(Edited by teerachild at 2:09 am on Feb. 8, 2001)


What happened when you increased the annealing temperature?