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Problem with PCR after bisulphite conversion
i have a problem with PCR. For a period of 4 months the methodology worked pretty fine, with no problems at all. Suddenly, PCR stopped giving visible bands. I have tried almost everything, new primers, new PCR Master mix, new MgCl2, lowering the annealing temp and still doesnt work. Its been a year since and i cant figure out whats wrong.
Re: Problem with PCR after bisulphite conversion
Is the template you are using still from the same and first batch? I noticed that the bisulfite treated template degrades over time, even while at -20deg C. It is a crude chemical conversion afterall, shearing most of your DNA anyway. PCR of bisulfite fragments can be troublesome, I use relatively long primers (22-25 bp) in nested setup and been able to produce >1kb fragments using regular taq this way. Touchdown PCR sometimes works to boost yield while keeping specificity. Using nested approach, template selection can be a problem, though for some templates it is unavoidable IMO. Ramp times are different for PCR machines, make sure you use the same machine and program you used earlier.
Last edited by Mathijs; 06-15-2009 at 09:55 AM.
|bisulphite , conversion , pcr , problem|
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