PCR Design

newsnet customer customer at newsnet.com
Mon Oct 9 03:59:41 EST 2006


Hi,

Two questions:

  1.. Is the Tm (melting temperature) of a primer calculated on the subsequence of the primer that binds to the template DNA? Why? because I have added restriction sites at the 5' end of the primer, which does not bind to the template DNA.
  2.. How is the Ta (annealing temperature) calcuated? from what I read, it should be 5 deg C less than the lowest Tm of the primer pair... but why?

Cheers,
ST


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