Pfu + Taq polymerase and poly-A tails - (Oct/15/2007 )
Hello All,
I am just curious if I use a mixture of Taq polymerase and Pfu Taq polymerase (6:1) for amplification, will the amplified product carry poly-A overhangs produced by Taq polymerase or they will be removed by exonuclease activity of Pfu?
Thanks in advance.
(I am planning to gel purify this PCR product and use it for TOPO-TA cloning)
Overhangs would be more likely removed.
I'd say, if you go for TA cloning the efficiency would be very low.
But if you have a short PCR product and big amount if it, there is a chance to clone it succesfully. You need only one good clone anyway.
Hi,
I would just use the Pfu polymerase in first place, and than add Taq (usually I use 1:8 ratio regarding Units) for additional 10 minutes at 72°C.
If you just have a specific PCR product you don't need to gel purify the product but use it diretly for TOPO-TA cloning.
Cheers
I'd go like bomber, have done this succesfully many times.
First of all, thank you everyone for chipping in.
I don't have specific PCR product, I get 3 bands and so I have to gel purify it. On the top of that the yield is pretty low and that is the only reason I thought of TA sub-cloning. That way, I would collect at least 5 different clones, mix them up and then clone it into the vector of my choice.
This PCR has been a nightmare for me. This PCR doesn't work at 56C annealing temperature but if I run a block on gradient PCR and set the tubes in 56C block then I get little product and I hope that this is of my interest. (I have cut out the band of expected size)
@Trof,
Why do you think TA cloning efficiency will be low? If I add poly-A overhangs like bomber suggested, it should be fine, right?
I don't have specific PCR product, I get 3 bands and so I have to gel purify it. On the top of that the yield is pretty low and that is the only reason I thought of TA sub-cloning. That way, I would collect at least 5 different clones, mix them up and then clone it into the vector of my choice.
This PCR has been a nightmare for me. This PCR doesn't work at 56C annealing temperature but if I run a block on gradient PCR and set the tubes in 56C block then I get little product and I hope that this is of my interest. (I have cut out the band of expected size)
@Trof,
Why do you think TA cloning efficiency will be low? If I add poly-A overhangs like bomber suggested, it should be fine, right?
for TA cloning I usually amplify first with Pfu, then gel purify, and then incubater my PCR product with dATP + Taq for 30 min at 72C. you can then use this straight away for ligation without further purification.
good luck!!!
can i ask you, why are you thinking of mixing 5 clones? Sure if you get your PCR in TOPO-TA or pGMET, you'll have enough product for further cloning from a miniprep.
PCR is not an error-proof way of DNA amplification, even if you use Pfu what if I select only one clone and it is messed up that I don't get product of my interest out of that sequence? So on the safe side, in my honest opinion, I would select 3 different clones and hope that at least one is fine but I select 5 clones because my boss wants me to select 5..
Even when I setup a PCR reaction, I do not setup one PCR of 50µl, I would setup 5 PCRs of 20µl each.
Hope this answers your question.
I have done a PCR with KlenTaq which contains a blend of Taq and a proofreading enzyme. Some A overhangs will be removed, but the cloning with the TOPO vector is quite efficient. I band purfied my products and successfully TA cloned them into the TOPO TA vector (pCR2.1).
I also use a comercial blend (Extensor enzyme mix, ABgene) and TA cloning works just fine. Just don't wait to long, purify your products within a few days of amplification.