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 Problem

Concentration ligation

  • Please log in to reply
4 replies to this topic

#1 alextam

alextam

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 02 February 2012 - 12:19 PM

Hi all,

I am recently having problems in ligating an insert into a vector. My vector is a 8kbp construct and my insert about 1,5 kbp. However after dephosphorylating my vector i have no colonies at all (Using T4 dna ligase). So I switched to the quick ligase kit, and they recommend to use 50ng of the vector and a 1:3 ratio for each insert. But is 50ng enough? As my vector is quite large, I am not sure whether 50ng of vector would be sufficient for the ligation reaction...

Thanks,

Alex

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,410 posts
234
Excellent

Posted 02 February 2012 - 01:18 PM

If you ligation is working well, then this is way more than enough.

#3 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 03 February 2012 - 12:44 PM

You will know when you try ligating with 50ng.

the manufacturer has probably tested dif. DNA conc. and is recommending 50ng.

#4 HOYAJM

HOYAJM

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 159 posts
12
Good

Posted 06 February 2012 - 11:57 AM

Promega recommends 100-200 ng of vector DNA for their T4 DNA Ligase. The NEB ligase recommends 50 ng. I think anywhere in these ranges should be fine. Also, try different ratios. In my latest clones, it was not the 1:3 that was optimal, but much higher (1:8)

#5 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 07 February 2012 - 08:54 AM

For cloning into some vectors, you might need to use a very high ratio.

With the NEB quick ligase kit, I follow the instructions and it works.





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

©1999-2013 Protocol Online, All rights reserved.