PCR trascendental questions

[DanielT]

I’m relatively a newbie in PCR but I’m trying to find out some info and didn’t get it.
I have these 2 concerns:

1) why is Taq polymerase so unstable at room temperature but stable at 95C?

2) All the PCR protocols I found mostly have a ratio of template/primers around 1:100. This is the problem I see: every cycle we do, we are consuming primers at the same rate of PCR products are formed. Basic maths showed that by cycle 6-7 (around 128 PCR products) all of our primers should have been used and therefore the PCR reaction shouldn’t go on.
I feel that I’m missing something important because all that I found repeat the 1:100 template/primer ratio.
I’ll appreciate your help!
Thank you,
David

[Daniel]

Firstly, Taq is not unstable at room temperature.

Secondly, I am not sure where you got this 1:100 template to primer ratio from but this is also wrong. I guess if you your assumptions are wrong then you should not be surprised when the results don’t make much sense 🙂

Daniel

Daniel Tillett
Nucleics Support

[Author]

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