Biparental Mating

Method:

1. Make overnight cultures of the following strains using appropriate antibiotics: E. coli C441 containing helper plasmid: Bacteria with the plasmid to be transferred, 30^oC. and P. aeruginosa recipient strain, 42^oC (ensure 42^oC water bath, as this temperature is required to shut off R/M system).
2. Place 0.1 ml of B in 2 ml of LB and add 0.1 ml of A.
3. Filter the mixture using a 0.45 um Nalgene filter unit. Remove the filter with sterile forceps and place filter side up on an LB agar. Incubate overnight at 30^oC.
4. Resuspend the bacteria in 2 ml of sterile saline (.9% NaCl). Make a 1:100 dilution of this suspension in saline. Spread 100 ul of the undiluted & diluted samples on selection plates. Grow up for 1-2 days at 37^oC.
5. Restreak colonies on selection plates to ensure purity and the presence of the antibiotic marker.