Promoter amplification.... - (Oct/08/2004 )
I am getting crazy at trying to amplify two different promoters that are not particularly GC rich (less than 60%). I tried with Taq, Pfu, Pfx, and Phusion enzyme and got nothing at all. And the template is a plasmid !! Any ideas about potential secondary structures in the template that could prevent amplification ??
Are you sure that you have used the right plasmid. If it is so hard to PCR amplify, then how do you make sure it is your target plasmid, by hybridization or just sequencing? and I think the sequencing procedure is also a problem as well as PCR.
I mean you must make sure that you have choose the right plasmid, primers will not anneal to the wrong template no matter how hard you've tried.
I'd agree. Run two sequencing reactions, one with each primer. You'll know that the primers are good and that your sequence is what you think it should be.
Actually, Now I have got something for one promoter I am trying to amplify, at the right size, but the band is very small and I have some by-product at a lower size. So I can reasonnably exclude that the problem is in the plasmid. And actually, this is not the first time that I have problems to amplify a promoter, but in the end, I always managed it by changing the enzyme or buffer. I am using primers with long tails (25-30 nucleotides that do not anneal) for Gateway cloning. I have never had this kind of problem when amplifying a coding sequence !!
Any other ideas ??
Try Expand PCR system from Roche.
The promoter region that i've been working on is also very AT rich and i've never had a problem. It could be worth making sure that your oligos are fairly long (>25?) in order to get decent Tm's.
Bioline also make a good polymerase called BioXact, which has worked for me in the past.
Both of these are high fidelity. I am using Expand for site directed mutagenesis of an AT rich region without any problems.
If you are worried about secondary structures, you could always linearise your plasmid template?
I never used Pfu enzyme. But this is the first time i will be using this Pfu to amplify promoter fragments of 2kb. I know pfu is very costly to use. so iwant to standerdize the conditions by using taq polymerase enzyme.Any body has an idea about the difference in annealing temparatures for Pfu and Taq polymerase.
Thanks a lot
Did you try AmpliTaq GOLD? works every time!
If you are sure that the plasmids are correct, just try to linearize them and PCR again. It has worked for me.