Hot-Start & Hi-Fi
p.r.ashby at dundee.MAPS.ac.uk
Wed Jan 17 12:20:52 EST 2001
In article <3a65cada$1 at news.unibe.ch>, "Bruno Cenni"
<bruno.removethis.cenni at removethis.insel.ch> wrote:
> Hi all,
> I am aware of polymerases that have proof-reading ability and others that
> come in a "Hot Start" flavor (chemically modified or antibody or others).
> there one that combines both? ...we are really concerned about
> errors... Has anyone ever tested if amplifying a stretch of DNA from a
> fairly abundant template (not single cells or such) with plain Taq
> introduces errors at such a rate (in the early cycles) that they'd be
> detectable by direct (ie. no subcloning!) sequencing of the PCR product?
Clontech do one of those, advantage HF2 PCR kit. Works well in my hands,
I optimise with taq then do it in anger with the clontech mix. I put
A-overhangs on the ends by adding a small amount of taq at the end
incubating at 72C for a couple of minutes then T-A clone the products.
I have no connection with clontech BTW.
Can't comment on the testing though the error rate of taq should be
online somewhere. I have used taq quite happily for directed mutagenesis
of up to 300bp without detectable error (other than intended) but I use
proofreading taq for cloning anything over 500bp.
Wellcome Trust Biocentre
University of Dundee
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