leaky hot start Taq polymerase

Roland Hubner 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.


Hi 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...

Cheers,
Roland




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