In response to Jonathan's initial question, you're not wrong - the
primers will denature before 94, but the full-length PCR product
you're trying to amplify is a lot longer and requires a higher melting
temp to dissociate.
also, if you're designing primers you can find some convenient code at
https://github.com/russelldurrett/Python_Primers
On Jan 27, 11:43 am, Jonathan Nesser <jonathan.nes...@gmail.com>
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
> diybioandneurosci.blogspot.com
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