Conjugation

Conjugation (on solid surfaces)

1.-Grow donor and recipient bacteria in LB broth with the appropiate antibiotics (overnight)

2.-Wash donor and recipient cultures in LB broth to remove antibiotics

3.-Mix 100 μL of donors + 100 μL recipients in an eppendorf tube (For a donor/recipient ratio of 1/1).

4.-Centrifuge briefly and discard supernatant.

5.-Resuspend the cell pellet in aprox. 10 μL of LB broth.

6.-Put a 0.22 μM filter (i.e Millipore filters) on top of a LB-Agar plate. Drop the conjugation mix on the filter.

7.-Incubate for 1 h (repetitive conjugation times are important) at 37ºC

8.-Pick the filter from the plate with sterilized tweezers and put it into an assay tube with 2 ml of LB broth

9.-Vortex to resuspend the cells

10.-Make serial dilutions in LB broth. Plate in selective antibiotics for donors, recipients and transconjugants. We usually determine donor number by plating 10^-4 10^-5 and 10^-6 dilutions. For transconjugants we routinely plate all dilutions (from 1 to 10^-6 ).

Modifications and tips

• Some plasmids require exponentially growing cultures to mate: add the following step: after step 1 dilute cells 1/20 in LB-broth and let them grow at 37ºC and orbital shaking. Check every 1/2 hour the OD₆₀₀ and collect the cells when the culture reaches 0.6 aprox.