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Pfu


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4 replies to this topic

#1 vetticus3

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Posted 24 November 2004 - 10:42 PM

Hi,
I'm supposed to be using Pfu to PCR-up some genomic DNA. I've used it before, it works... most of the time. Has anyone had problems with the enzyme dying on them? I'd like to think that my last dozen PCRs haven't worked because I'm incompetent, not the enzyme... but it hasn't worked for the other assistants in the lab either.
Is there another alternative that's more reliable?
Thankyou.
Vetticus

#2 Simonsays

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Posted 25 November 2004 - 05:46 AM

Try Taq polymerase...

Simon

#3 tuckern

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Posted 25 November 2004 - 09:27 AM

The whole point of using PFU etc is that it's a proof reader. Ordinary Taq will incorporate lots of errors in your PCR product - our lab uses it for random mutagenesis, so i would advise against this accuracy is important to you.
I never touch Pfu - having been mentally scarred by it. It just didnt happen for me. I changed to another high fidelity enzyme and it worked first time - no errors.
I think it was Bioline's bio x-act that worked in the end. PWO polymerase is also good.

#4 Janina

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Posted 26 November 2004 - 12:16 AM

Did you check the buffer? It has to be with MgSO4...

#5 Charon

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Posted 28 November 2004 - 10:11 AM

Well, overall I found Pfu to be less stable than Taq. It may be best to aliquot the polymerase instead of risking the whole vial.
Another factor is that Pfu works slower than Taq, but as you used it already sucessfully I suppose that this was already considered.




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