"CpG methylation: Blocked by some combinations of overlapping" (View forum version)


Posted 21 August 2011 - 11:19 AM

I am looking to perform a double digestion with PacI and NheI. NEB tells me that NheI is affected by some combinations of methylation. My plan is to PCR amplify an ORF from cDNA with restriction site compatible ends and clone it into a plasmid grown up in DH5a cells. How do I know if methylation will affect the NheI digestion on the plasmid?



Posted 21 August 2011 - 12:24 PM

NheI is not affected by methylases present in E. coli lab strains. CG methylation is common in mammalian DNA, and might be an issue if you were digesting with NheI directly from isolated eukaryotic DNA. If your DNA has been cloned in E. coli, there should not be any issue.