Hello Tina,

Thank you for your answer. The problem is we dont know if the DNA is methylated or not, so using it as a control for methylation specific primers is not useful. I am aware of the uracil conversion, and thus designed primers that were supposed to bind the DNA even after treating it with bisulfate.The problem is that even my Negative Controls NC are positive. I have tried several primers so far and no luck.

i advise you to give it another shot with different extraction kit..as i had an awful experiments with Zymo products..try Qiagen or Thermo

good luck

Dear Heba,

Thanks for your reply, I am indeed using Qiagen kit for DNA extraction, but for the bisulfate treatment I am using Zymo Research.

I think i sort of solved the problem, by extracing new DNA, and using Platinium polymerase. It could be that the DNA I had was fragmented or whatever.

Effectively, the using of Platinum Polymerase can solve the problem . I solved a similar problem to testing several Polymerase such as the Platinum Polymerase, the EpiTaq HS (Takara) and the KAPA2G Robust HotStart (KAPA BIOSYSTEMS).

We use everything Zymo and it's been good so far. Both the extraction/conversion kit and the polymerase. You need uracil-tolerant polymerase and degenerate primers for BS-treated DNA to amplify.

I am having the same problems with my DNAg bisulfite-converted sequences. I´m not able to amplify BS-treated. Yoann Menon did you tryed with kapa2G robust and you could only amplify by using platinum polymerase?

Yes, finally I used preferentially EpiTaq HS and the KAPA2G Robust polymerases to amplifiy gDNA BS-treated. I obtained better results with these pol compared to Platinum

In our hands using nested or heminested PCRs highly improve the results, even when in our first round of PCR we don't see any band. The addition of DMSO clearly improve the results too