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Female
Hi everybody,
I have been wondering and searching but haven't found a method that enables distinguishing the paternal and maternal allele with all the modifications and proteins bound to DNA. This would be extremely helpful when studying imprinted genes and their regulatory proteins and modifications - now bisulfite seq and ChIP only give a mixed result.
Separating the alleles could be possible at the chromosome level, but how? Any ideas or comments? I have heard about some technique that uses mitosis as a starting point but the regulatory mechanisms cannot be studied with the same strategy. I have thought that artificial triplex structures in the area of many maternal/paternal SNPs could serve as a starting point for chromosome capture but applying this technique might be impossible.
Don't hesitate to share your ideas or comments, I'm looking forward to them!
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Bisulfite PCR doesn't work and the primer seems abnormal
20 September 2012 - 02:58 PM
Hello,
I have tried to solve this problem but now my supervisor can no longer help!
I have the primers and the program from the publication and they should be ok.The only change is that we added restriction sites to 5' ends of the primers so they are 36 and 39 nt in length, and we use Phusion Hot Start polymerase instead of Hot star taq.
Kuva0535.jpg 93.6K
73 downloads
I have tried two different bisulfite conversion programs and still don't get any result. We thought something would be wrong with the primers so I measured them with Nanodrop and the other showed an abnormal curve (only one side of a hill with two slopes) and 260/280 of 2,2 and 260/230 of 0,8. A newly synthesised primer shows the same curve and values. Is it possible that inpurity interferes with PCR reaction? Is it possible that this polymerase can't achieve to make about 600 bp of product?
What would you do in my shoes? I'm happy to hear some suggestions and advice!
I have tried to solve this problem but now my supervisor can no longer help!
I have the primers and the program from the publication and they should be ok.The only change is that we added restriction sites to 5' ends of the primers so they are 36 and 39 nt in length, and we use Phusion Hot Start polymerase instead of Hot star taq.
Kuva0535.jpg 93.6K
73 downloadsI have tried two different bisulfite conversion programs and still don't get any result. We thought something would be wrong with the primers so I measured them with Nanodrop and the other showed an abnormal curve (only one side of a hill with two slopes) and 260/280 of 2,2 and 260/230 of 0,8. A newly synthesised primer shows the same curve and values. Is it possible that inpurity interferes with PCR reaction? Is it possible that this polymerase can't achieve to make about 600 bp of product?
What would you do in my shoes? I'm happy to hear some suggestions and advice!
