Thanks for the hint with the Clontech enzyme!
For my other question: I found plenty of references for the error rate
measured by analyzing clones after PCR. I am not sure how these number
translate in probability to introduce an error in the first cycle and to do
that in a sufficiently high frequency (same mutation) with several molecules
of template present in at least ng amounts such that this mutation would be
visible in direct sequencing of the PCR product (ie. to get about 50 % of
the product with a mutation).
"Peter Ashby" <p.r.ashby at dundee.MAPS.ac.uk> wrote in message
news:p.r.ashby-D9040C.17205217012001 at dux.dundee.ac.uk...
> 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
> > come in a "Hot Start" flavor (chemically modified or antibody or
> > 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 Ashby
> Wellcome Trust Biocentre
> University of Dundee
> Dundee, Scotland
> Reverse the spam and remove to email me.