Routine PCR isn't working - (Sep/27/2007 )
I am PCRing up 4.2kb region. I started off with Qiagen Long Range PCR kit (for fidelity issues and thats only what we had in the lab). I didn't get any PCR product. As usual, I thought of wrong annealing temperature so I ran a gradient PCR from 50-60C, got decent profile and realized that I ran previous PCR at right annealing temperature.
I get pretty fat band using normal Fermentas Taq polymerase but at the same reaction conditions, I don't get anything using Qiagen kit. I tried once more using different primer stock (don't ask me why...) No PCR product.
Today, I tried PCR with Fermentas Taq polymerase and Fermentas Pfu Taq polymerase and WTF, no band again....
I am not getting a clue what is going wrong here. Anybody else apart from me getting suspicious that proofreading is creating any issues? If I use only Taq as in gradient PCR, I get fat bands but if I use Pfu Taq/Qiagen Taq (with proofreading activity), no PCR product
Any suggestions are appreciated.
Thank you in advance.
Hmm... could you run some of your template on a gel. As a rule of the thumb the first suspect when anything goes wrong is template degradation.
I haven't run the template on the gel but its the same template used for gradient PCR and the others.
I am also not clear about something. The gradient PCR, was it conducted using your proof reading polymerase or plain Taq? Different polymerases work best under different conditions (extention temperature, time) using their own specially formulated buffers.
The proof reading polymerase don't amplify as fast as Taq. It can be a low as 1/10 the speed. This could explain why you see a signal with Taq and nothing with Pfu and other proof reading polymerases. (And is the Taq actually amplifying 4.2kb? Could I have your conditions for this, please? That is a rather big product for plain Taq. I wonder what you do to get it to go that far.)
Have you tried anything else, (increasing Mg ion concentration, BSA)
Could you list the PCR conditions and the PCR reaction mix. It would help.