Can you use longer incubation time with XhoI to Overcome CpG methylation?

Alexander Supertramp @Alexander_Supertramp3

07 July 2019 1 2K Report

I've purified my plasmid from DH5a for a double digestion (NcoI and XhoI), however I've noticed it only appears to be cleaving at one site (ie I'm only getting one band in the gel). Previously it worked with plasmids purfied from Top10 cells - I think because they can prevent CpG methylation, whereas DH5a does not. NEB says XhoI is CpG methylation impaired but I don't know what that actually means with regards to digest efficiency or speed. so can I just increase the digestion time?

Thanks

Michael J. Benedik

You should not be getting CpG methylation from DNA isolated from bacteria. So I don't think this is the problem. XhoI should not be sensitive to bacterial methylation patterns.

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