Conjugation on filters
Inoculate small ( 2 or 3 ml ) cultures of your donor and recipient strains , with appropriate antibiotics , in lysogeny broth ( LB ) .
Do this late in the day , the day before you want to do the conjugation .
Take 1 ml of donor and recipient culture and centrifuge at 10,000 rcf for 2 minutes to pellet the bacteria .
Aspirate and discard the supernatant and resuspend the bacterial pellet in fresh LB with no antibiotics .
First thing in the morning on the day of the conjugation , place sterilised 0.2 um pore size filters on an L - agar plate without antibiotics .
Use aseptic technique .
You will need one filter for each conjugation you're setting up , plus one for each strain on its own as a control .
Pipette 10 ul of the resuspended donor and recipient strains in the same place on a filter , sitting on the agar plate .
Do the same for each strain on its own as a control .
Incubate the plate upside down ( agar suface facing up ) at 37 degrees Celsius .
Near the end of your work day , take the plate out of the incubator .
Using aseptic technique , pick each filter off the plate using forceps and place it in a 1.5 ml tube containing 1 ml of sterile PBS .
Vortex the tubes to free the bacteria from the filter .
Remove and discard the filters from the tubes using aseptic technique .
Plate desired volumes of the bacterial suspension on selective agar medium and incubate over night .
