PCR Design

[newsnet customer]

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