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 conjugation failed

pseudomonas conjugation e.coli s-17

  • Please log in to reply
1 reply to this topic

#1 thefallengrace

thefallengrace

    member

  • Active Members
  • Pip
  • 17 posts
2
Neutral

Posted 16 January 2014 - 12:58 AM

Hi there,

 

im doing conjugation between pseudomonas with e.coli s-17, harbouring a gene replacement vector with OriR6K origin.

 

Here is the protocol i used:

 

1.grow both recipient and donor for 10 hours

2. mix 100ul of each bacteria, and drop 200 uL on LA, incubate at 37 overnight.

3.took a loopful of the bacteria mixture, and resuspend in 1 mL sterile ddH2O

4. spread 100 uL of it on LA + 100 Amp(kill e.coli) + 25 Gm (kill pseudomonas)

5. incubate at 37c for 48 hours

 

 

and i got no colonies AT ALL??, negative plate have no colonies. 

 

 

what have i done wrong?

i thought conjugation should be easy?

maybe i should grow the starter culture overnight and concentrate it before i mate them?



#2 vallie

vallie

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 17 September 2014 - 05:54 PM

Does your vector have gentamycin resistance? If not, then your Pseudomonas won't grow. Also, you may try incubating the plates a little longer (although 48 hrs should be enough for pseudomonas, I believe). Bacteria grow slower in the presence of antibiotics. Are you sure your pseudomonas is ampicillin resistant?







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

©1999-2013 Protocol Online, All rights reserved.