https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide

Hi Sedef,

I came across this issue and often it was due to the low concentration of DNA used initially, which was the further reduced by DNA degradation in the conversion reaction. This issue was particularly prevalent when bisulfite converting DNA from FFPE tissues. In order to overcome this I tried a number of different bisulfite reaction kits and PCR set-ups, and in the end used this kit: https://www.zymoresearch.com/epigenetics/dna-methylation/bisulfite-conversion/ez-dna-methylation-lightning-kit

I still had this issue occasionally but much more consistent results with this kit as it seemed to be more gentle on the DNA.

Hope this is helpful!

Thank you for your answer however I am currently using this conversion kit you mentioned above. However I am still getting the same results. Have you ever tried visualizing your bisulfite converted DNA on agarose gel and succeed ?

Hi Sedef,

Yep I was able to visualise on agarose except in the cases of some particularly degraded samples. I did have to carry out a lot of optimisation of the PCR steps however, and I would use a very thin 2% agarose gel. I suggest you try a number of different annealing temperatures using the control samples and go from there.

I hope this helps!