generally annealing temperatures will be just 3-4 C below Tm of primers. if the melting point of primers is very high like you said in the range of 90 , at that hight tempertaure all double stranded dna will split off and so annealing will not take place., because annealing happens only in the range of 50 - 65 C. hope i'm correct.. if i'm wrong anyone can correct me...
Jonathan Nesser wrote:
Sorry to start yet another new thread for such a small question, but I
don't want to pollute other threads with off topic posts... I've been
trying to understand why primer melting temperatures have to be in the
55-65C range when PCR cycles call for 94C for DNA melting... I've
tried to find an explanation for this but haven't been able to...
Wouldn't that result in the primers splitting off from the template
DNA before you reach the extension cycle of 75C (or around there)? I'm
sure I'm wrong because obviously these PCR programs work, but I don't
know WHY I'm wrong, and would like to understand. Thanks again for
explaining something that is probably so common knowledge that nobody
feels the need to address it in writing :)
Jonathan Nesser
jonathan.nesser@gmail.com <mailto:jonathan.nesser@gmail.com>
diybioandneurosci.blogspot.com <http://diybioandneurosci.blogspot.com>
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