Hot-Start & Hi-Fi

[Peter Ashby]

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). 
> Is
> there one that combines both? ...we are really concerned about 
> introducing
> 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.

Peter

-- 
Peter Ashby
Wellcome Trust Biocentre
University of Dundee
Dundee, Scotland
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