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!
Photo
- - - - -

ligation


  • Please log in to reply
1 reply to this topic

#1 priyak

priyak

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 26 March 2009 - 07:40 AM

hi team

i just started working with cloning....my problem is that i did n't get any transformed colonies my vector is pcr2.1 of 4000bp, and my insert size is 1620bp, digested with Kpn1 and Bamh1. i checked the efficiency of my competent cell,its fine,checked the efficiency of ligase enzyme.its fine

Then i icreased the insert:vector ratio from 1:3,1:5, increased incubation..1hr to overnight. but no colonies....can any one help me in this problem?


Thanks
priyak

Edited by priyak, 26 March 2009 - 07:41 AM.

pk

#2 rkay447

rkay447

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 177 posts
20
Excellent

Posted 26 March 2009 - 08:11 AM

hi team

i just started working with cloning....my problem is that i did n't get any transformed colonies my vector is pcr2.1 of 4000bp, and my insert size is 1620bp, digested with Kpn1 and Bamh1. i checked the efficiency of my competent cell,its fine,checked the efficiency of ligase enzyme.its fine

Then i icreased the insert:vector ratio from 1:3,1:5, increased incubation..1hr to overnight. but no colonies....can any one help me in this problem?


Thanks
priyak

Your ratio is the wrong way. You want a 3:1 insert:vector ratio. This alone could be the reason why you aren't getting colonies. You need to make sure your quantification is correct by running a small amount of the insert and vector together on a gel. Compare intensity to a quantification marker. Take 25ng vector and, according to the sizes you said a 3:1 ratio with 25ng vector would mean you need to add 30.4ng insert. Reset up the ligation with the proper insert ratios and try it again.




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

©1999-2013 Protocol Online, All rights reserved.