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 with cloning p16ink4a into pEGFP-C2

  • Please log in to reply
1 reply to this topic

#1 kijitengu



  • Members
  • Pip
  • 1 posts

Posted 12 April 2011 - 11:40 PM

Hi everyone!
I do cloning p16ink4a into the pEGFP-C2. I digested them with HindIII and SalI in seperate reactions. After that I ligated the vector and insert DNA in ratio of 1ul:3ul; 1ul:1ul; 3ul:1ul (recommendation of Promega); 1V:3V (V vector/V insert) and 1m:3m (m vector/m insert) and set up ligation using T4DNA ligase (16 degrees, 16 hrs). Then I did the transformation into E.coli DH5α. I got a few of colonies on 1ul:3ul; 1ul:1ul; 3ul:1ul plates (A) and many colonies on 2 remaining plates ( B ).
To test colonies, i do PCR-colony with the primers are C-EGFP and SalI. Majority of B have the band at about 540 bps. I pick up the colonies which have bold band and shoot the tip into the tube within 5ml Kanamycin. The recombinant plasmids were extracted by Plasmid mini-prep kit (Solgent Co., ltd) and test by PCR with the primers are C-EGFP and SalI. But i got no band. I test again by PCR colony with the colonies on backup plate and the result is no band I re-do this process many time but the results do not change.
Please give me some advice!!!
Thanks a lot!

Edited by kijitengu, 12 April 2011 - 11:41 PM.

#2 bob1


    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,511 posts

Posted 14 April 2011 - 08:46 PM

The presence of the insert in the transformation mix may be giving your false positives when you pick and PCR off the plate.

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

©1999-2013 Protocol Online, All rights reserved.