Me again...on type of polymerase and REs I should use... - (Nov/15/2005 )
ok its me again.... 
....here im trying to design PRC experiment with primer that contain restriction enzymes termini...and after that my cDNA (PCR product ) will be ligated into another vector (GST) ....so i have some sites for RE on that vector but best would be near the N terminus so they are EcoRI and XbaI ....but i have read that EcoRI shouldnt be used cause it has star activity 
and XbaI has been reported to have troubles in cutting DNA close to ends....but according to New England biolabs catalog their effeciency would be 100% and 99% so....should I use them?
another question is simple...would taq polymerase do fine in my PCR (if there are restriction sites) or I should look for another one??
thank you so much..... 
have a great day!
....here im trying to design PRC experiment with primer that contain restriction enzymes termini...and after that my cDNA (PCR product ) will be ligated into another vector (GST) ....so i have some sites for RE on that vector but best would be near the N terminus so they are EcoRI and XbaI ....but i have read that EcoRI shouldnt be used cause it has star activity and XbaI has been reported to have troubles in cutting DNA close to ends....but according to New England biolabs catalog their effeciency would be 100% and 99% so....should I use them?another question is simple...would taq polymerase do fine in my PCR (if there are restriction sites) or I should look for another one??
thank you so much.....
have a great day!Hi.
Regarding your choice of polymerase to use for your cloning, I would not recommend taq as this might not copy your target faithfully. I use KOD hotstart from Novagen, this has much greater fidelity, is super fast at elongation, and of course is hotstart, which minimises unspecific amplification. Pfu from Promega is also good. Hope this helps.
hi
i've succesfully used EcoRI in its dedicated buffer overnight without getting star activity. If you are afraid about that you can perform your digestion in a PCR tube by a simple program : 37° for 4h and then "hold" 4°...
for xba1 you'll get only 10% of digestion in the EcoRI buffer. So you'll need to do a double digest.
Except if you do a digestion in buffer 2 + BSA. 100% of activity (supposed) of EcoRI and XbaI
and i've done it overnight whithout star activity (you can either choose a pcr facility if you want)
For amplification : up to 1.5kb i use Taq polymerase and it"s ok. in cases of i want 100% secure i sequence DNA. And if i can't, i use proof reading polymerases. But i've successfully cloned 1Kb Taqamplified whithout any mistake.
i've succesfully used EcoRI in its dedicated buffer overnight without getting star activity. If you are afraid about that you can perform your digestion in a PCR tube by a simple program : 37° for 4h and then "hold" 4°...
for xba1 you'll get only 10% of digestion in the EcoRI buffer. So you'll need to do a double digest.
Except if you do a digestion in buffer 2 + BSA. 100% of activity (supposed) of EcoRI and XbaI
and i've done it overnight whithout star activity (you can either choose a pcr facility if you want)
For amplification : up to 1.5kb i use Taq polymerase and it"s ok. in cases of i want 100% secure i sequence DNA. And if i can't, i use proof reading polymerases. But i've successfully cloned 1Kb Taqamplified whithout any mistake.
thanx a lot everyone...my protein is 1.7 Kb so do think Taq will still be ok?
phusion polymerase from NEB is also high-fidelity (although not hot-start) and is much cheaper than KOD
good luck
thanx everyone im checking the polymerases you have suggested....plaese i would like to learn more about type of polymerases everyone is using in their PCRs.....Special case: my protein is 1.7 kb and im adding RE termini to my PCR product.....thanx a lot again! have a great day everyone
I generally find that I can amplify a PCR product of up to 3 or 4 kb easily with either regular Taq or a proof-reading (high fidelity) variant. My choice of one versus the other is determined by what I intend to do with the amplified DNA -- if I'm going to clone the product, I'll use a high-fidelity polymerase, if I'm just screening transformants or something, and just need a yeah-nay from the PCR reaction, I'll use regualr Taq.