Erickson:Tri-parental Mating

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(New page: Tri-parental Mating 1. 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) 2. Combine 200 µL of...)
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Revision as of 20:01, 20 May 2008

Tri-parental Mating 1. 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) 2. Combine 200 µL of the donor, helper, and recipient strains into one centrifuge tube 3. Centrifuge and remove supernatant 4. Re-suspend pellet in 25 µL broth 5. Spot onto BHI plate (no antibiotics) 6. Incubate overnight at 30°C 7. Scrape a colony from plate and re-suspend in broth 8. Dilute each solution to 10^-1 9. Plate 100 uL of each solution onto a BHI plate with kanamycin and irgasan 10. Incubate overnight at 30°C

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