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?