- Grow ON of yeast in SD 2% glucose + 25 µg/ml EtBr
- Culture a second ON from 1st ON, in SD 2% glucose + 25 µg/ml EtBr
- Plate for single colonies on YPD plate (let grow 2-3 days 30C)
- every clone should be rho0 (colonies should be small/slow)
- verify no growth on YPG and slower growth on YPD
- bioscreen in YPD media and look for slower Dt vs WT and no diauxic shift
- back cross with WT strain to compliment YPG growth deficiency
- verify by DAPI staining using log phase cells
- grow cells to mid log (OD ~0.4-0.6) in SD 2% (if possible, avoid YPD, since it gives lot of background?)
- Add DAPI ( 1 mg/ml stock) to the final concentration of 2.5 µg/m
- Grow the cells for 30 min w/DAPI
- spin down cells, wash with 1X PBS,resuspend the cells in PBS,
- perform microscopy (use WT and known rho0 cells as controls)
DAPI WT vs rho0