Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
- - - - -

"CpG methylation: Blocked by some combinations of overlapping"

  • Please log in to reply
1 reply to this topic

#1 BSM



  • Active Members
  • Pip
  • 15 posts

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?


Edited by BSM, 21 August 2011 - 11:20 AM.

#2 phage434



  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,747 posts

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.

Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.