no pcr product with pfu/pwo but with taq - (Feb/28/2006 )

hello!

I'm trying to amplify a 2.1kb insert from a pET101D/TOPO vector, which I obtained from a Midi-prep.

In order to find the optimal annealing temperature for my primers, I performed some gradient PCRs with several polymerases.

Interestingly, the reaction worked with Pfu resp. Pwo polymerase and T7 primers (that recognize the T7 promoter priming site resp. the T7 reverse priming site within the vector), but not with my self-designed primers.

But when I used Taq polymerase, I obtained a product with the self-designed primers too.

I tried this several times with different conditions (different annealing temperature, different elongation times,...), but I cannot amplify my insert with my primers AND Pfu/Pwo polymerase.

I need the product for some cloning reactions, therefore I have to use a proofreading polymerase.

Please can anybody give me some suggestions on this case?

Thank you in advance!

Try very low annealing temperatures, or try higher Mg-concentration. Maybe try another proofreading enzyme (pfuturbo or pfuultra, pfx, pfx50, Vent/deep Vent, phusion...) or a mix of a proofreading enzyme with taq (expand high fidelity, platinum taq, ...)

It seems that the primers you designed are some how at fault. Whether it's an issue with the sequence or reaction conditions is not clear.
Taq, without proofreading ability, will some times overlook improper annealing of primers making it less descriminate in what it will amplify.

Look at your primer sequences and make sure they are within the template sequence. If you can resequence you plasmid prep to make sure the sequence you're working with is the actual sequence of the plasmid (some sort of rearragment or mutation could have occured within the 'primer picking' regions).
Good Luck

Thanks for your advices!

I tried stepdown-pcr and pcr with lower annealing temperatures. I added extra MgSO4 too and increased annealing time for Pwo-polymerase.

Unfortunately it did not work (neither with Pwo, nor with Taq-polymerase). I suppose the primers don't fit the template DNA exactly. Perhaps there are silent mutations in my protein.

Tomorrow I am going to send my template to the sequencing lab...