leaky hot start Taq polymerase
roland.hubner at ua.ac.be
Mon Jan 21 12:18:02 EST 2002
> Dear All,
> I spent years using hot start, then last year stopped doing it
> (because I couldn't find the %$@$ing option for freezing the PCR program
> of the new PCR machine). Nothing really bad happened to my reactions. I
> have been using PCR on plasmid DNA to to cloning.
> I assumed that a cold start was OK because all the template was locked
> away as dsDNA and could not intereact with primers before the first
> denaturation step. Is hot start only really important for low
> abundance targets in complex mixtures, eg genomic DNA?
> Regards, Mike.
whether a hotstart will improve a given PCR can be tested quite easily
by omitting Mg++ from the reaction & adding that at high temps... (no
need for PAUSE button)
If you obtain an improvement and have a lot of samples to do, avoiding
contamination, trying to accurately quantitate rxn's etc., then you
start getting interested in automagic activation methods...
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