leaky hot start Taq polymerase
mw132 at mole.bio.cam.ac.uk
Mon Jan 21 11:39:51 EST 2002
On Mon, 21 Jan 2002, Roland Hubner wrote:
> > using platinum hot start polymerase I got all the
> > same lots of primer dimers products, is it anybody else experience too?
> > does anybody has a better source to suggest? what about the
> > chemically modified ones, are they more expensive too?
> Hello Vuvu!
> ==> "Rockstart" buffer is _EXCELLENT_ ( http://www.klentaq.com/ ); you
> can drop the initial denaturation step completely... good for longish or
> long-range amplifications...
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?
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