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!
- - - - -

Self ligation of vector

  • Please log in to reply
No replies to this topic

#1 milki



  • Members
  • Pip
  • 4 posts

Posted 08 April 2010 - 09:45 PM


I am facing a problem in clonning of 45 bp insert in 3.5 kb plasmid. Actually i am getting self ligation of vector rather than with insert ligation. I run a control (only vector) parallel with the ligation of insert and the result with control is 2-3 colonies while with insert 10-12 colonies. From these 10-12 colonies only three are clonned with insert.

I have gone through following reasons.

1. Problem with dephosphorylation of vector (i did it again)
2. I varied the concentration of insert.
3. I varied the concentration of vector.
4. I have checked the several combinations of insert and vector ratio (1:3 to 3:1).
5. Vector concentration is 10-15 ng in per ligation reaction.

The main problem is that i am getting more colonies with self ligated vector in the plate where insert was supposed to ligate while the control where no insert was present i usually get 2-3 colonies. I know that it is normal because covalent linkage is made within the bacetria by DNA repair mechanisms.


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

©1999-2013 Protocol Online, All rights reserved.