Erickson:Tri-parental Mating

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  • 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