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PCR primers with overhangs - (Oct/03/2007 )

Hey you all!

I´ve designed primers with overhangs in their 5´end, and the question I asked myself is that when I make the PCR, di I have to change the Tm after the first cycle. I mean since in the first round the Tm is different from the second depending on a smaller size /a part of the primer that attaches.
Can you help me with this problem?

Thank U.


In principle, perhaps. In practice, I never do this and don't have a problem.


No you don't. And like phage434, neither do I in practice. the annealing temperature that i use is based only on the tm of segment of the primer that actually binds the template.

Instead think of it as a natural touchdown PCR. As more of PCR product forms, the effective tm of the primer increases (as the 5'end is now included) but as the annealing temperature has not changed, the specificity is decreased slight and your get more product.