Erickson:Tri-parental Mating
From OpenWetWare
Jump to navigationJump to search
- 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
