TRIPARENTAL MATING (1)

REFERENCE: Goldberg and Ohman. (1984). J. of Bacteriol., 158:115-1121.
 

METHOD:

  1. Make overnight cultures of the following strains using the appropriate antibiotics:
    1. Bacteria with the plasmid to be transferred, 30ºC.
    2. E. coli C199 containing the helper plasmid pRK2013, 30ºC with 25 µg/ml kanamycin.
    3. P. aeruginosa strain which will be the recipient, 42ºC.
  1. Place 0.1ml of C into 2ml of L-Broth, then add 0.1ml of A and B.
  2. Filter this bacterial mixture using a 0.45 um Nalgene filter unit. Remove the filter and place the filter cell side up on an LB agar. Incubate the plate overnight at 30ºC.
  3. Resuspend the bacteria from the plate in 2 to 5 ml of sterile saline. Make a 1/100 dilution of this suspension in saline. Spread 100 µl of undiluted and 1/100 dilutions on selection plates. Grow 1 to 2 days at 37ºC or until distinct colonies are seen.
  4. Restreak colonies on selection plates to ensure purity and presence of antibiotic marker.





Updated 01 December 2000.