Grow yeast cells in YPD medium at 30° C to mid-log phase.
Pellet cells in 50 mL in blue cap tubes and spin at 3500 rpm for 5 minutes at RT° (room temperature). Use the Labofuge 400 with swinging bucket rotor.
Resuspend in 1.7 mL potassium phosphate/sorbitol buffer (10 mM potassium phosphate/ 1.2M sorbitol pH 7.2)
Add 5.1 µL b-mercaptoethanol (bME) and 544 µL lyticase (1 mg/mL stock solution made in water).
Place in shaker for 15 minutes at 30° C.
Pellet cells by spinning at 5000 rpm in the Beckman J2-21 using the JA-20 rotor. RT°
Wash 1X in 2 mL potassium potassium phosphate/sorbitol buffer. Pellet cells at 5K rpm in the Beckman J2-21 using the JA-20 rotor. RT°
Remove supernatant, add 1 mL H2O but do not disturb the pellet. Spin 5K rpm in the Beckman J2-21 using the JA-20 rotor at RT°. Use a pasteur pipet to remove the liquid and save cells.
Add 500 µL H2O and resuspend cells until no chunks are visible.
Freeze thaw in -70° C three times in a row. (> 20 minutes each time)
Follow DNeasy kit beginning with step number 2 on page 15. Use 50 µL of the frozen cell gamish for your gDNA extraction.
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