Annealing temperature for PCR - (Aug/10/2004 )
I have a forward primer 74 bps (Tm is 70 ) and reverse primer 24 bp (Tm 61). Can you suggest me how I should calculate anealing temperature.
As thumb of rule, you can use an annealing temperature that is 5-10 below the Tm.
Here is an article discussing this issue. You can get it from Pubmedcentral free
Thanks for your reply.
Even if i subtract 5-10 from tm...then should I subtract from tm of forward or reverse primer?
normally I'd subtract from the Tm of smaller primer. If you are worried about the specificity of your reaction you can do a test run with a few different annelaing temperatures