(by tk at mit.edu)
Mon Oct 9 09:38:12 EST 2006
"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
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