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!
- - - - -

Ligation of double insert into pET31 failing

pet31 double insert

  • Please log in to reply
1 reply to this topic

#1 nathanley



  • Members
  • Pip
  • 1 posts

Posted 24 November 2011 - 02:56 AM


I would appreaciate any advice here, it feels like I've been banging my head against a brick wall for weeks now.

I am trying to clone an 81 bp fragment (in two halves with a 4 bp overlap) into pET31, however after ligation and transformation I get ZERO colonies.

I digest pET31 with AlwNI, dephosphorylate and then gel extract the linearised vector. This is fine. After this however I can't get my insert to anneal. I anneal the two halves separately and then ligate all together with the vector in the same reaction. I have tried ligation overnight at 4', on the bench for 2h at room temp, and have altered most of the parameters I.e. reduce the amount of ligation mixture (either 5 or 10 uL) per 60 uL of competent cells.

Does anyone have any ideas?


#2 phage434



  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,747 posts

Posted 24 November 2011 - 05:00 PM

Do your oligos have a 5' phosphate? If not, that would explain the problem. You can add them with a PNK treatment before or after annealing.

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

©1999-2013 Protocol Online, All rights reserved.