Help with making positive control - SssI methylase treatment and then? (Jul/30/2008 )
I am preparing a postivie control for CpG methylated mouse genomic DNA.
I treated SssI with mouse genomic DNA purified manually.
Reaction condition like this:
SssI methylase 10U (2.5uL)
SAM 320 uM
NEB buffer #2
water to total 50uL
4hrs at 37'C
I'd like to confirm if this DNA can be used as a globally CpG methylated positive control.
Can anybody help me how I could break through here?
Its going to be difficult to assess if its globally methylated. You could try Bisulfite sequencing on an area that was not previously methylated (GAPDH would be a good gene to use) and look for full methylation. You could also use a restriction enzyme digest, but probably not as good as some bisultfite sequencing.
Is it for use as a control for bisulfite sequencing? The only other thing I can suggest is New England Biolabs, who once sold methylated DNA, but they would have used SssI methylase to obtain it.
Our lab has historically treated with sssI methylase twice to ensure that we get 100% methylated DNA ie after 4hrs re-add the enzyme.
Also one thing to make sure of is that the SAM is new, it goes off quite quickly i believe!
Davo and Frozenlyse, thanks so much for your help!
This is for MSP, and I've never tried bisulfite sequencing.
I thinking about the best ways to set up experimets for DNA mehtylaion studies in my lab.
Commercial methylated DNA seems to be a good start, and after that I can also try with SssI further on.