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

E. coli growing in LB-ampicillin medium without plasmid? Or miniprep problem?

E. coli selection plasmid

  • Please log in to reply
2 replies to this topic

#1 Paperdog

Paperdog

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 04 March 2014 - 05:11 AM

I received an E. coli miniculture (not frozen, cooled with a cooling bag) sent by an international collaborator. (One day long travelling)

The day I received it, I inoculated into LB-Amp medium and then next day I made a STET and a Wizard miniprep. Both contained the correct plasmid. (Checked by test digest and agarose gel electrophoresis.)

The same day I made a glycerol stock. Just measured glycerol and E. coli together, vortex and quickly to -80°C. (15% glycerol)

 

Two days later I inoculated this glycerol stock. It grew in LB-ampicillin, but after STET miniprep it contained no plasmid. How is it possible? The ampicillin is stored at -20°C, it shouldn't degrade.

The same day I retransformed the first STET prep into another E. coli strain and next day it grew on the plate, but the colonies were very small.

 

My questions are:

- is it possible that an E. coli culture grows in Amp without selection plasmid?

- what can be the problem in the process (in the original strain, in the glyc culture, in the STET prep? )



#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,468 posts
247
Excellent

Posted 04 March 2014 - 05:40 AM

I would never receive a sample this way. You should always streak such a sample on a plate, pick a single colony, and prepare a glycerol from a liquid culture grown from that colony. Somewhere across the ocean or later some of your cells lost the plasmid. This is easy, because ampicillin resistant colonies release lactamase which degrades ampicillin, and eventually there is little left. Then, when you grow up a new culture, the cells that have lost the plasmid out-compete the plasmid containing cells. Fortunately, all you have to do is retransform the plasmid you have, and things will be fine. I'd recommend switching to carbenicillin from ampicillin (more stable), and raising the amount to 100 ug/ml to reduce the problem of growth without a plasmid present.



#3 lyok

lyok

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 206 posts
6
Neutral

Posted 05 March 2014 - 01:32 PM

I would never receive a sample this way. You should always streak such a sample on a plate, pick a single colony, and prepare a glycerol from a liquid culture grown from that colony. Somewhere across the ocean or later some of your cells lost the plasmid. This is easy, because ampicillin resistant colonies release lactamase which degrades ampicillin, and eventually there is little left. Then, when you grow up a new culture, the cells that have lost the plasmid out-compete the plasmid containing cells. Fortunately, all you have to do is retransform the plasmid you have, and things will be fine. I'd recommend switching to carbenicillin from ampicillin (more stable), and raising the amount to 100 ug/ml to reduce the problem of growth without a plasmid present.

I have been looking into buying carbenicillin because the ampicillin gives too much problems.

However in the lab where I work they dont want to buy it because its very expensive.


Now I am looking into where to buy it and I found this:

The companies where we buy our stuff ask 297dollar (http://www.sigmaaldrich.com/catalog/product/sial/c1389?lang=en&region=BE) or http://www.lifetechnologies.com/order/catalog/product/10177012 300 dollar.

 

How is this possible?

Is this 46 dollar product ok?







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

©1999-2013 Protocol Online, All rights reserved.