Erickson:Tri-parental Mating


 * Tri-parental Mating

Grow the donor, helper, and recipient strains of bacteria separately in 3 mL of LB Broth overnight (30° C for Y. pestis and 37°C for E. coli) Combine 200 µL of the donor, helper, and recipient strains into one centrifuge tube Centrifuge and remove supernatant Re-suspend pellet in 25 µL broth Spot onto BHI plate (no antibiotics) Incubate overnight at 30°C Scrape a colony from plate and re-suspend in broth Dilute each solution to 10^-1 Plate 100 uL of each solution onto a BHI plate with kanamycin and irgasan Incubate overnight at 30°C