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Annealing temperature for PCR


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3 replies to this topic

#1 sve02594

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Posted 10 August 2004 - 07:43 PM

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

#2 labrat

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Posted 10 August 2004 - 08:56 PM

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

#3 sve02594

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Posted 10 August 2004 - 09:22 PM

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

#4 kant0008

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Posted 10 August 2004 - 09:36 PM

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




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