need a whole genome methylation protocol - (Jan/07/2008 )

Hi there.

I need to validate some results and therefore want to make a set of standards with known methylation levels. Given that I work with deer samples I haven't been able to purchace universally methylated/unmethylated DNA.

I am assuming that if I use a standard WGA kit I can get unmethylated DNA, but could someone provide me with a protocol for methylating genomic DNA?

Thanks

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Hi,
if you simply treat your DNA with SssI methylase you will end up with fully methylated DNA. The CpG Methyltransferase, M.SssI, methylates all cytosine residues (C 5 ) within the double-stranded dinucleotide recognition sequence 5'...CG...3. This works fine, we had to make some controls a while ago and ended up doing this (that way you can make a set of standards with known levels of methylation). We also use universal available controls as well.

krizzy

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And the protocol is on the NEB website :-) Do add some extra SAM and Sss1 after your initial round (we do two times 4 hours of incubation) as it will improve the methylation %.

Bram

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