Quick note here
Annealing temp needs to be just low enough for primers to re-associate
with complementary strands. This is based on affinity, so, as a
previous poster pointed out, primer association % is in a state of
equilibrium.
Dissociation / Denaturing / Melting (whatever you want to call it)
tempurature is dependent on the bond strength between the template
strands, and the energy needed to dislodge Taq. The high (94 degree)
temp ensures that G-C (triple bond) rich regions are thoroughly
split. double-stranded DNA is super stable, so once the double helix
is formed (post elongation), you really need to blast the system with
heat energy before those bonds start to break.
On Jan 31, 10:36 am, Nathan McCorkle <nmz...@gmail.com> wrote:
> I think its wrong to say annealing only happens between 50-65 C, but
> that's the general range for most primers 15-25 bp in length
>
>
>
>
>
> On Fri, Jan 27, 2012 at 11:57 AM, veera <drveeramanikan...@gmail.com> wrote:
> > 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.nes...@gmail.com <mailto:jonathan.nes...@gmail.com>
> > diybioandneurosci.blogspot.com <http://diybioandneurosci.blogspot.com>
>
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> Nathan McCorkle
> Rochester Institute of Technology
> College of Science, Biotechnology/Bioinformatics- Hide quoted text -
>
> - Show quoted text -
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