Liquid Mating Assay

1. Dilute O/N 1:100.

2. Let grow at 30°C, shaking to an OD₆₀₀=0.5. (With a 1:100 dilution it usually takes 4-4.5hrs)

3. Spin cells for 5¢ at 2000rpm.

4. Resuspend cells in enough LB so that you can mix the cells in a 1:5-10 ratio; DY330 F¢ donor: TB1.

5. Let sit at 30°C for 90¢, no shaking.

6. Vortex for 10s. to break apart stable mating pairs.

7. Incubate on ice for 10¢.

8. Spin cells at 4000 rpm for 8¢ in cold room centrifuge; aspirate.

9. Resuspend in 1ml Sterile 1X PBS.

10. Make serial dilutions (1:10², 1:10⁴, 1:10⁶)

11. Plate 100l onto LB strep-tet-chlor and M9 Biotin-tet-chlor. (Plate DY330 on LB-strep-tet, no chlor)

12. Also, plate 100l onto LB-strep to make sure TB1 cells are OK.