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
- - - - -

Trouble with growth of transformed cells


  • Please log in to reply
4 replies to this topic

#1 Linda Ferreira

Linda Ferreira

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 18 February 2011 - 10:50 PM

Hi there

I am a Biotechnology student busy with a techniques course. We are doing cloning. Everything has been going well, but for some reason my cells transformed with my recombinant plasmid DNA, as well as those transformed with the ligation control, refuse to grow.

I know my cells are competent as my positive control and all the subsequent dilutions grew just fine. The plates were also made from the same batch of LB broth, amp, tet, IPTG and X-gal. I also have recombinants - did an agarose gel analysis (cloned 1200bp fragment into linearised pBS, EcoRI used as restriction enzyme)

I really don't know what is going on. Did the same procedure twice now, with no growth, blue or white, on those two plates.
Any suggestions will be sincerely appreciated

Regards
Linda

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,475 posts
251
Excellent

Posted 19 February 2011 - 06:29 AM

Is your positive control a transformation of plasmid, or is it cells already containing the plasmid?

#3 Linda Ferreira

Linda Ferreira

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 19 February 2011 - 09:14 AM

It is a transformation of the pBS plasmid isolated through large scale purification.

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,475 posts
251
Excellent

Posted 19 February 2011 - 11:25 AM

OK, you should be able to calculate the transformation efficiency of your cells by counting colonies on dilutions of your plasmid. You should get 10^7 to 10^8 colonies per microgram of plasmid DNA if you have good efficiency. Less than that makes it difficult to ligate and then transform. Many other things can go wrong, but usually it is either poor transformation efficiency or poorly prepared DNA going into the ligation reaction.

#5 Linda Ferreira

Linda Ferreira

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 22 February 2011 - 11:08 PM

Thanks for the advice. Calculated the transformation eff and it was quite low.
So, I managed to increase the efficiency and I got a few colonies! So yes, thanks a lot!




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

©1999-2013 Protocol Online, All rights reserved.