How can I determine the efficiency of bisulfite conversion? Who has the detailed protocol?

I use the Qiagen EpiTect Fast DNA Bisulfite Kit to do bisulfite conversion. However, I fail to clone any target fragment (for 6 genes). I can't find the reason.

Could you help me? Thank you very much.

Hi Yanfeng,

I really do not know quantitative methods for assessment bisulfite conversion.

For more information see: https://www.researchgate.net/post/How_can_I_quantify_Cytosine_Uracil_in_DNA_using_bioanalyzer_Agilent#view=56ea7a1beeae3902e14cd79a.

Vasiliy V Reshetnikov

Hi, Vasiliy. Thank you.

The bisulfite conversion is going to fragment your DNA and leave single stranded DNA behind which makes the nanodrop and similar methods lacking. From what I've read based of the Zymo kits is using gel electrophoresis to assess it. Probably the most cost effective way outside of sequencing the region. Its an awkward thing to do in routine labs.

From my own experience with Zymo kits, 99% conversion efficiency is what they claim with a >75% DNA recovery from the original input amount. I say your best bet is to go with their stated claims unless you have time to kill verifying it but I would include a control gere. For example take a positive control, put it through the same temperature cycles with everything bar the CT conversion reagent and process it with you primers and probes geared towards your methylated genes but also have a primer and probe that is geared towards the non converted DNA regions. This would allow the control DNA that was converted to swap the cytosine residues and not bind to the primer and probe and the non-converted DNA to bind and produce a detectable product. Should be negative in the first instance and positive in the second indicating the conversion worked.