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Pfu polymerase mixed with Taq polymerase

Pfu polymerase mixed with Taq polymerase - Protocols and Methods Forum

Pfu polymerase mixed with Taq polymerase - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 05-24-2006, 11:39 AM
Julio Cortazar
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Default Pfu polymerase mixed with Taq polymerase



Hi there,

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


Jacob

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  #2  
Old 05-24-2006, 06:32 PM
Nick Theodorakis
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Default Pfu polymerase mixed with Taq polymerase


Julio Cortazar wrote:

I believe that is similar to the original Long Accurate PCR protocol by
Wayne Barnes. You may find this discussion helpful:

<http://www-lecb.ncifcrf.gov/~pnh/papers/TIBS/aug94.html>

Nick

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  #3  
Old 05-24-2006, 07:14 PM
Austin P. So (Hae-Jin)
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Default Pfu polymerase mixed with Taq polymerase

Julio Cortazar wrote:


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.

Austin

P.S. another thing you may consider is getting your overlapping primers
(?) purified...at least find out what the purity of your primers are.
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  #4  
Old 05-25-2006, 02:22 AM
ChenHA
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Default Pfu polymerase mixed with Taq polymerase

On 24 May 2006 04:39:07 -0700, "Julio Cortazar" <[Only registered users see links. ]>
wrote:


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
worked.

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.




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  #5  
Old 05-26-2006, 09:03 AM
Duncan Clark
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Default Pfu polymerase mixed with Taq polymerase

Historians believe that in newspost
<1148470747.430100.317820@j33g2000cwa.googlegroups .com> on Wed, 24 May
2006, Julio Cortazar <[Only registered users see links. ]> penned the following literary
masterpiece:

There is a Polish manufacturer/supplier of Pwo (100% identical to Pfu)
so should not be too expensive.

DNA-Gdansk II

[Only registered users see links. ]


Duncan
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I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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  #6  
Old 05-26-2006, 05:07 PM
Julio Cortazar
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Default Pfu polymerase mixed with Taq polymerase

Thanks Duncan! Soon I will call that team

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  #7  
Old 05-27-2006, 11:59 AM
newsnet customer
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Default Pfu polymerase mixed with Taq polymerase

> >I come from a country where less that 1% of country earnings is
[Only registered users see links. ]
ds=9758761&dopt=Abstract

Invitrogen sells HiFi Platinum Taq that has both high specificity and
fidelity.

[Only registered users see links. ]
etails&productDescription=512&CMP=LEC-GCMSSEARCH&HQS=11304%2D029

Sharp Tool



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