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  • 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)
Rho0

DAPI WT vs rho0

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