Pwo PCR

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Mon Mar 9 12:41:48 EST 1998


In article <6dontn$m0d at mserv1.dl.ac.uk>, Nico Dantuma
<nico.dantuma at mtc.ki.se> wrote:

> Hi,
> 
> I have some problems amplifying fragments using Pwo polymerase. I know that
> this enzyme is more demanding than ordinary Taq. Still I would like to use
> it because of the blunt ends of the fragments. Even when I use plasmid
> template getting a product is very difficult. There is not much to be
> optimized because there is no product at all. What is the best way to
> optimize the reactions when you know the optimal conditions for ordinary
> Taq? (more/less template, more/less cycles, how much do you have to lower
> the annealing temperature etc.) Is this blunt end cloning of fragments that
> have been amplified with polymerases with proofreading commonly practized?
> With other words: does it work? I need it for constructing fusions so I
> have to be absolutely sure that the frame is correct.
> 
> Thanks in advance,
> 
> Nico
> 
> 
> ______________________
> Nico Dantuma
> Microbiology and Tumorbiology Center
> Karolinska Institute
> Box 280
> S-171 77 Stockholm
> SWEDEN
> Phone: +46 8 7286280; fax: +46 8 330498

Hi Nico,
If you know the conditions for Taq, double the extension time (all othe
parameters can be left unchanged). In the case of Pfu polymerase (my
favorite one!), use at least 2min per 1000bp. Routinely, I allow 150sec
for extension per 1000bp, and it works perfectly.
Hope this helps,
Frank



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