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
- - - - -

Bacteria grew on plates but not in liquid culture


  • Please log in to reply
1 reply to this topic

#1 JHW

JHW

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 26 July 2013 - 08:48 AM

I was doing simple ligation to get my PCR-amplified insert into pGEX vector to express recombinant protein. After ligation, I screened colonies by colony PCR and inoculated positives clones into amp-containing LB for overnight. However, none of them grew. I tried it twice, used different batches of cut vectors, freshly made new plates and still, I got colonies on plates but no growth once inoculated into liquid culture. In the beginning, I suspected it might be the ligation efficiency (e.g. all clones I picked were false positive), or ampicillin was too old, etc. However, it was not the case because I did the following experiments.

I took the empty vector, amp-resistant pGEX, and transformed it into XL1-blue competent cells. At the same time, I used non-transformed XL1-blue competent cells as control. I plated 120 uL non-transformed (NT) and transformed (T) cells into LB agar plates (+/-amp) or inoculated 2 uL directly into LB medium (+/-amp or carb). Here are the results:

NT cells:
1. LB plate -amp: grew and covered the whole plate
2. old plate (~1 week) +amp: none grew
3. new plate +amp: none grew
4. LB medium -amp: grew, turbid
5. LB medium +amp from our lab: none grew
6. LB medium +amp from lab A: none grew
7. LB medium +carb from lab B: none grew


T cells:
1. LB plate -amp: grew and covered the whole plate
2. old plate (~1 week) +amp: many single colonies
3. new plate +amp: many single colonies
4. LB medium -amp: grew, turbid
*5. LB medium +amp from our lab: none grew
*6. LB medium +amp from lab A: none grew
*7. LB medium +carb from lab B: none grew

As you can see, * were the problems, I got colonies on the plates, but the same vector-transformed cells couldn't grow in liquid culture. Does anyone have suggestion what the problem might be?

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,512 posts
371
Excellent

Posted 27 July 2013 - 01:32 PM

Two potential problems could be - 1, false +ve from the colony PCR (often due to picking up DNA from the transformation mix that has been spread on the plate) and 2, a toxic insert that will support some growth for a limited period of time.






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

©1999-2013 Protocol Online, All rights reserved.