M.SssI treatment of DNA - (Feb/23/2009 )
This is my first post and I'm not sure that I've done this correctly, but I have a question about methylation... I want to use M.SssI to treat 10ug of genomic DNA and I'm not sure what volumes to use for the M.SssI, NEB Buffer2, and SAM and how long I should let it incubate?? The NEB protocal gives volumes for 1-4ug of DNA, but I can't find anything for 10ug. Thanks!
M.SssI treatment of 10 µg DNA shouldn't be such a big problem. The NEB protocol can be upscaled. But the concentration of SAM should be increased from the standard concentration (160 µM) up to.. well, this depends on your DNA-type, density of CpGs, etc... At the least I would double the concentration of SAM. Incubate your DNA for 2-4h at 37°C. For complete methylation add fresh methylase and SAM and incubate overnight.
Hope that helps...