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.

Thanks

Sandy

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
http://www.pubmedcentral.nih.gov/articlere...ubmedid=2243783

Thanks for your reply.
Even if i subtract 5-10 from tm...then should I subtract from tm of forward or reverse primer?

Thanks

Sandy

Hi,
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