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!
- - - - -

Restriction double digest


  • Please log in to reply
1 reply to this topic

#1 Ramiz Sheikh

Ramiz Sheikh


  • Members
  • Pip
  • 1 posts

Posted 07 December 2014 - 04:31 AM

Hello everyone,


I want to subclone my gene into pet22b vector. I did double digestion of both vector and insert plasmid with Nde1 and HindIII 1ul each in 50ul reaction with buffer 2 and incubated for 3 hours at 37degree.  Also as controls i digested my samples with individual enzymes also. After agarose, i found out that the vector was linearized and the band was moved upward than the uncut vector. The problem is with my insert plasmid. With individual enzymes, i am getting a single band of linearized plasmid. However, with double digestion, 3 bands are seen which concludes that the plasmid has not been digested. Everytime i get different results with double digestion. i think enzymes are working fine.. Can any1 please shed some light on this?? Thank you

#2 phage434



  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,747 posts

Posted 07 December 2014 - 06:47 AM

What is your setup for the restriction digest that is not working? (How much volume of each of the components)

A common problem is inhibition by impurities in the minipreps of plasmid DNA. This happens most often when the major volume of the digestion is dilute DNA solutions, rather than water.

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

©1999-2013 Protocol Online, All rights reserved.