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!
- - - - -

Problem restriction digest on single fibers

restriction digest

  • Please log in to reply
No replies to this topic

#1 KimV



  • Members
  • Pip
  • 3 posts

Posted 23 May 2012 - 10:59 AM

I'm trying to perform a restriction digest on single fibers. I'm using 2 enzymes BanII and Cviki-1. What I do is I cut 1 fiber out of muscle tissue on a slide (LCM) and perform than a dna extraction. Then i do a PCR with a fam labbeled primer. In the next step I do the restriction digest. The problem I experience is that not the whole sample is digested. about 10% stays undigested. I tried already using different restriction buffers, adding more enzyme, longer incubation times, adding BSA but nothing seems to help.
If I start with 200 ng DNA in a pcr reaction and perform than the restriction digest everything is digested. So I assume it has something to do with the small amount of DNA.
Does anybody have experience with restrction digest starting from small amounts of DNA?

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

©1999-2013 Protocol Online, All rights reserved.