PCR and pH of buffer

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Tue Feb 8 11:17:30 EST 2000


Mark Denhart wrote:

> Does anyone have an opinon on PCR and the pH of the buffer?
>
> I've been wondering recently, how or why this would affect the
> reaction.  I've heard reports from friends that some primer/template
> combinations work better at pH 9.0, others at pH 8.3.
>
> It must be a very complex aspect of PCR - since you have TRIS (which you
> normally pH at 25C), pH is affected by an increase or decrease in
> temperature....
>
> Thanks for your input!
>
> Mark
>
> ---

Hi Mark,
I guess most of the people who use PCR in research work agree that there is
quite a lot of voodoo involved. That´s to say: you do something and hope it
has the right effect. In some cases it will, in others it won´t. I don´t
want to say that PCR is generally independent of pH, but the problem is
that you cannot predict which PCR will work better at higher or lower pH.
So, it is up to you to check it. I personally like to change parameters
like Mg concentration, annealing temperature, additives like DMSO,
formamide or betaine, rather than trying pHs from 7 to 9 in 0.1
increments....  But, if you are desparate and have tested everything else,
pH titration might do the job for you. Just remember that the very next
time it  may not have any effect.

And, yes, of course, pH in the final reaction goes up and down with cycling
from annealing to denaturation because of buffering with Tris.

Frank






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