Bacteria conjugation failed - (Jan/16/2014 )

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?

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?