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
- - - - -

1-2kb PCR products TOPO cloning troubles


  • Please log in to reply
4 replies to this topic

#1 julie

julie

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 30 September 2004 - 11:49 PM

Well here's my problem:

I'm trying to clone 3 differents PCR products into PCR4-TOPO. Their length vary between 1.3 kb and 2.5 kb, wich is not that huge I think.
The thing is I used Pfx (proofread) to amplify the fragments, then added the A overhangs by adding dNTP and Taq + 10 min 72C. I then used the max amount of PCR products to perform the ligation reaction, and so forth : I made everything advised to get results, but it doesn't work!!!

Should I purify the PCR products after adding the As? Should I use more vector in the ligation reaction?
Should I...? I don't know what to do!!!

If you know how to solve my problem please tell me...
Thank you very much

#2 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
68
Excellent

Posted 01 October 2004 - 04:29 AM

Hi Julie,

Have you included any controls in your experiment? Controls are essential for ligation and transformation troubleshooting as has discussed here many times. You can use intact plasmid such as pUC which is usually included in cloning kits as positive control. If you could not see colonies in control transformation, the problem may then be related to your competent cells.

I think you should purify your PCR reaction before ligation.

How about including a Taq amplified PCR product as a control for the effciency of adding a "A" by the Taq following Pfx amplfication?

#3 julie

julie

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 03 October 2004 - 10:12 PM

Hello and thank you fo your help!

As a matter of fact I made some control reaction, ie I cloned another plasmid (=> my competent cells are ok); and I couldn't clone the fragment after Taq amplification either (ok this I should have told sooner... :) ).
So I'm a little desperate! But I'm gonna try purifying the fragment before ligation (and maybe saying a little prayer or something!?!)

Thanx again!

#4 julie

julie

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 04 October 2004 - 10:19 PM

:) It works!! Petri dishes full of bacteria!!
Thanx again and again!!

#5 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
68
Excellent

Posted 04 October 2004 - 10:41 PM

congratulations!

Actually I often spend a sleepless night after doing a transformation and fear I may not see those lovely and shiny colonies the next morning.




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

©1999-2013 Protocol Online, All rights reserved.