Pfx PCR problem - (Jun/15/2006 )
If I use Taq polymerase I am able to amplify my cDNA sequences. But when I try to use the same cDNA samples and same primers I am unable to obtain a product using the proofreading Pfx polymerase (Invitrogen).
I need to amplify a 1200bp and 800 bp fragments. I have confirmed cDNA is good by using Taq polymerase and tubulin´s primers as positive control.
Does anyone have any ideas of what may be the problem?
Have you tried to use another cDNA as template for your proofreading Taq? If it doesn't even amplify another cDNA, probably the Taq doesn't work at all. If it does, I don't know what could be wrong... Good luck!
I was in a similar situation a while ago. Taq polymerase would amplify but another polymerase from NEB, wouldnt amplify at all. Never knew what was happening.
A suggestion from invitrogen is to use double concentrated buffer. It has worked consitently better for me.
Have you considered playing around with the annealing temp? Buffers are different in salt concentrations, it might influence Tm of your primers...
I use Taq poly to amplifying the fragments and next I use the product as templeted to the Pfx.....but only one product I have been able to amplify. Thx for all...... I will attempt your suggestion...
I have exactly the same problem - PCR on cDNA works fine with normal Taq but not proof-reading. Spoke to Invitrogen yesterday and they reckoned its because I still have some uracils in my DNA which the proofreading taq can't deal with (says so in the guidelines) but the ordinary one can because of 3- to 5' exonulcease activity or something. They recommend that you use UDG when making the cDNA or when doing the PCRs
I routinely had this problem with normal Taq amplifying easily and Pfx having trouble. The only that ever worked for me with Pfx was when I was using a plasmid as a template. The High fidelity Taq polymerase which is a combo of polymerases seems to work well and the mutation rate is not as high as with Taq. I now use this when trying to clone something mutation free from cDNA samples.