PCR Design

[Tom Knight]

"newsnet customer" <customer at newsnet.com> writes:

>   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.

The first cycle involves only the section binding to the template.
Once amplification starts, the full primer length binds.

>   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?

I use a simple formula for the annealing temperature of my PCR
reactions: Ta = 55C.  This almost always works if you have reasonable
primer designs.  People worry FAR too much about what the Tm of their
primers are.  Only rarely can a poor primer be rescued by changing the
annealing temperature.  Use Primer3 to design your primers if you have
problems.