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Quantifying % conversion with SsI methytransferase ? - how quantify the amount of C's converted in amplicon of interest (Aug/16/2007 )

I have an amplicon of 1.5kb that i want to fully methylate with SssI methyltransferase. I can do this no problem, but i need the conversion to be over 95% of the C's, and i need to have a VERY precise measurement to ensure convserion.

Bis sequencing is incomplete as not all mC's are converted.

What are my options?

-sneth-

If I understood you correctly, you need to be sure that >=95% of Cs in CG's are methylated by M.SssI?
Then you may try to use tritium labelled AdoMet and compare tritium incorporation by M.SssI into non-methylated and completely methylated (M.SssI + non-radioactive AdoMet) DNA.

-CH3-

How many times do you sss1-treat your amplicon. It may be necessary to have 2 or more rounds for full methylation.
But I am still a little confused - do you need to be sure to have a 100% conversion by bisulfite treatment or full methylation of CpG-C's by Sss1?

-kr├╝melmonster-