QuikChange II Site-Directed Mutagenesis

Duncan Clark blackhole at abuse.plus.com
Tue Jan 10 08:09:10 EST 2006


Historians believe that in newspost <dpudl4$12g$2 at news.doit.wisc.edu> on 
Mon, 9 Jan 2006, D.K. <no.email at thanks.to.spam.net> penned the following 
literary masterpiece:
>In article <QbZu8JDFdiwDFAFH at abuse.plus.com>, Duncan Clark <news at genesysltd.co.uk> wrote:
>>Historians believe that in newspost <dpt86c$au5$1 at news.net.uni-c.dk> on
>>Mon, 9 Jan 2006, Jens Mollerup <jmollerupREMOVE at REMOVEmy.molbio.ku.dk>
>>penned the following literary masterpiece:
>>>>        Of course, Stratagene does not really tell what ligase they add
>>>> and what substrate it takes - which allows them to raise the price
>>>> of the kit by 1.5X. Anyone knows of any commercially available
>>>> thermostable DNA ligase?
>>>>
>>
>>It will probably be Pfu Ligase using ATP as cofactor as opposed to Taq
>>ligase using NAD as cofactor.
>
>Why do you think so?
>.

' Cos Stratagene sell Pfu Ligase for which they have a patent - 
US5506137 or WO9402615

Fidelity is supposed to be better over the Taq ligase.

 From observation, Stratagene will use their own IP wherever possible.

>I'd imagine the choice comes down to
>1) polymerase/ligase buffer compatibility, 2) substrate stability
>(NAD vs ATP). I am assuming that both enzymes are similarly
>termostable and similarly active..

However although the patent (EP1417327 or WO03025118) for the multi-site 
mutagenesis does claim for Pfu Ligase (amongst others), the main 
examples for the actual suggested final kit use Taq ligase, Turbo Pfu 
and FEN1 (presumably Pfu FEN). Turbo Pfo being Pfu plus Pfu dUTPase.

So probably the best homemade would be Phusion plus a thermostable 
dUTPase plus a FEN plus Taq ligase and NAD.

I personally would go for Phusion over Pfu 'cos in my hands it works 
much better than Pfu. IMHO the Sac7 domain really improves polymerase 
enzymes - MJ patents plus papers.

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

Duncan Clark
GeneSys Ltd.


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