Mix Pfu and Taq - whether it can be done (Jan/17/2006 )
My friend suggested me to mix Pfu and Taq DNA polymerase to amplify gene for cloning. That means Taq for polymerisation and Pfu for proofreading. But I doubt whether it can be done. Please give me your idea!
A lot of the commercial "taq high fidelity mixes" (expand high fidelity, platinum taq high fidelity, ....) consist of a proofreading enzyme combined with taq, so yes, it can be done. But, be carefull concerning the buffer needs of your different enzymes, and also, the ration of Pfu to Taq will also be important...
Will work, but be careful if you want to to TA cloning because Pfu generates blunt ends. Appropriate volume of Taq and Pfu will make it.
Could you explain for 'buffer needs' and 'the ration of Pfu to Taq' clearly?
I started using this setup recently for routine cloning and stuff. I use a 10:1 ratio of taq to PFU in my PCR reactions. I use the buffer supplied with the Taq. I found an article where they tested a bunch of different buffers in like the Biotechniques journal or something and they found that the Taq buffer was the best. I tried to dig up the article for you but I lost it sorry!
PFU is ridiculously expensive! The system seems to work alright and saves on cash. I used it to PCR up 1kb amplicons, which I cloned, and none of the inserts were mutated. That being said, I didn't bother doing a Taq only amplification to compare. At 1kb there may not have been many/any mutations anyway.
It is quite common to mix both enzymes for high fidelity plus good proof reading. Haven't used it yet by myself as there are other polymerases here for my stuff - but from a paper I dug out just for that matter in preparation of possible needs the mix they used was 1.25 Units Taq Polymerase + 0.15 Units Pfu (stratagene).