cDNA cloning using Taq/pfu polymerase - (Nov/12/2007 )
Hi, I have a basic cloning question
I have seen and read that almost everyone uses Taq polymerase to clone a gene from a cDNA isoalted by RT-PCR from RNA. My question is can we also use a proof reading polymerase like Pfu to clone??????or does Taq has its own advantages???????
Taq produces too many errors but is robust and amplifies targets well. Pfu has proof-reading activity so it produces very few errors but is not very robust and does not amplify targets that well. Phusion polymerase is a protein fusion of Taq and Pfu. It has the proof-reading activity of Pfu but also the robustness of Taq - it is the best polymerase to use for cloning. It's not expensive, I wouldn't use anything else.
Moreover, if you're using a proof reading polymerase, it doesn't leave A-overhangs which you can latter use for TA-cloning, like Taq does. But what people do in these cases is a ~20min incubation of the amplified products with Taq, so you can get the A-overhangs.