Conjugation
Conjugation (on solid surfaces)
Grow donor and recipient bacteria in LB broth with the appropiate antibiotics (overnight)
Wash donor and recipient cultures in LB broth to remove antibiotics
Mix 100 μL of donors + 100 μL recipients in an eppendorf tube (For a donor/recipient ratio of 1/1).
Centrifuge briefly and discard supernatant.
Resuspend the cell pellet in aprox. 10 μL of LB broth.
Put a 0.22 μM filter (i.e Millipore filters) on top of a LB-Agar plate. Drop the conjugation mix on the filter.
Incubate for 1 h (repetitive conjugation times are important) at 37ºC
Pick the filter from the plate with sterilized tweezers and put it into an assay tube with 2 ml of LB broth
Vortex to resuspend the cells
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.