3 replies to this topic

[sve02594]

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

[labrat]

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.pubmedcen...ubmedid=2243783

[sve02594]

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

[kant0008]

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