Have heared that one may mix Pfu and Taq polymerases (Fermantas). Taq
is copying Pfu verifying. Have any of u tried this combbination? I'm
planning to introduce two nucleotide changes in 1kb DNA fragment. So
far I made set of 6 PCRs (I'm using so called SOE-PCR method with
overlapping extensions) but my RedTaq polymerase made so many mistakes
that I started to sworn it very badly.
Thanks for advise
I come from a country where less that 1% of country earnings is
subscribed for science. We do not have money for reactions using only
Pfu polymerase, so that's why I asking u these things
I remember looking into this, and if my memory serves me correct, a 1:9
mixture of Pfu to Taq is used for analysis of single nucleotide
polymorphisms by an HPLC based technique. I don't know if anyone has
tried it for longer templates though.
That being said, I don't remember ever finding a unit concentration that
people used, or what people used to dilute the polymerase mixtures.
P.S. another thing you may consider is getting your overlapping primers
(?) purified...at least find out what the purity of your primers are.
On 24 May 2006 04:39:07 -0700, "Julio Cortazar" <[Only registered users see links. ]>
Have you tried KOD polymerase? It seems much cheaper than Pfu and
probably work just as well. It probably worked out much cheaper
because you using the Pfu polymerase you use 2.5U but Kod use 1U. I
tried it for gene synthesis and it worked when Pfu failed. I haven't
tried it for mutagenesis yet, but others in the lab have tried it
using Quikchange protocol substituting Pfu with KOD and said that it
It also come with dNTP which is a bonus. Personally I wouldn't use
Taq for anything other than just seeing if something can be amplify
especially when everything else failed. Cloning/mutagenesis I
normally use high fidelity enzymes.
Historians believe that in newspost
<firstname.lastname@example.org .com> on Wed, 24 May
2006, Julio Cortazar <[Only registered users see links. ]> penned the following literary
There is a Polish manufacturer/supplier of Pwo (100% identical to Pfu)
so should not be too expensive.
[Only registered users see links. ]
I love deadlines. I especially like the whooshing noise they make as
they go flying by.