Erickson: Bi-parental Mating

1. Grow up the separate cultures in the selective media overnight:

    BO81 in BHI + Strep
    B628 in BHI + Km + Cm
    B629 in BHI + Km + Cm
    Y. pestis KIM6+ in BHI + Irg
    Y. pestis ymt in BHI + Irg

2. Pellet and resuspend the pellets in 200 uL plain BHI. 3. Combine the separate cultures and sperad on plain LB plates.

    25 uL B628 + 75uL BO81
    25 uL B629 + 75uL BO81
    25 uL B628 + 75uL ymt
    25 uL B629 + 75uL ymt
    25 uL B628 + 75uL KIM6+
    25 uL B629 + 75uL KIM6+    

4. Grow overnight at 30 degrees Celsius.

5. Resuspend the lawn in 1.5 mL BHI broth.

6. Perform serial dilutions of 0.1, 0.01, and 0.001.

7. Plate 100 uL of each suspension and its dilutions on selective plates. BO81 cultures on Km + Srep and Yersinia cultures on Km + Irg.

8. Grow overnight.

9. Count colonies.