Taq polymerase works but Phusion polymerase doesn't (View forum version)



lotus

Posted 11 August 2010 - 11:23 AM

I am trying to amplify an 800 bp product from a pooled mix of crude C.elegans lysates. The primers are as published in an old paper and they work great with taq polymerase (NEB). Unfortunately, I am getting a lot of SNPs when I sequeneced about 10 clones derived from the PCR product. I don't know if these mutations are introduced by Taq or are already present in the pooled worms. So I want to re-do the PCr using a high-fidelity polymerase. I tried Phusion (Finnzymes) which has always worked fabulously whenever I have used it. But now, I just can't get any band. I use the protocol that comes with the enzyme. I have tried HF buffer and GC buffer with 3% DMSO. All I get is non-specific, very faint low-size bands.
How do I marry the efficiency of taq pol. with the fidelity I am looking for?

HomeBrew

Posted 11 August 2010 - 02:12 PM

Try a high fidelity Taq -- we use this one. I just got sequence back from a ~1000 bp clone I made this week, and it was perfect.

perneseblue

Posted 11 August 2010 - 08:05 PM

Try a high fidelity Taq -- we use this one. I just got sequence back from a ~1000 bp clone I made this week, and it was perfect.


Urg... I got an error message. HomeBrew, could you write down the name of this product? Thanks!

HomeBrew

Posted 12 August 2010 - 04:19 AM

Sorry -- it's Platinum Taq DNA Polymerase High Fidelity from Invitrogen (cat. no. 11304-011). Try this link. It's a mixture of Taq polymerase and the proofreading Pyrococcus species GB-D polymerase, and supposedly provides six times higher fidelity than Taq alone. To be perfectly accurate, we actually use the "SuperMix" form of this enzyme (Platinum PCR SuperMix High Fidelity, cat. no. 12532-016, see here).