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-   -   Problem with PCR after bisulphite conversion (http://www.molecularstation.com/forum/epigenetics-forum-dna-methylation-histone-chromatin-study/65118-problem-pcr-after-bisulphite-conversion.html)

Spyridonid 03-11-2009 03:59 PM

Problem with PCR after bisulphite conversion
 
Hello
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.
Any ideas?

:cry:

Mathijs 06-14-2009 11:59 PM

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.


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