• Prepare 50mL of fresh YPD in an Erlenmeyer flask (volume?).

• Start an overnight culture and let the cells grow for about 15h (for strain CEN.PK113-5D) in order for the cells to reach the stationary phase.

• Harvest the cells in a 50 mL falcon tube and centrifuge at 3000g for 5 min. at 4ºC.

• Wash the cells in ice-cold 0.9% NaCl (twice).

• Ressuspend the cells in 1 mL of NaCl 0.9% and transfer the suspension to a 2mL Eppendorf tube.

• Centrifuge the cells at 3000g for 5min and discard supernatant.

• Add 1mL (total volume) of desintegration buffer.

DISINTEGRATION BUFFER:

Compound	Concentration	Stock Solution	for 50mL
TEA (triethanolamine) pH 7.0	100 mM	1 M	25 mL
EDTA pH 7.0	0.5 mM	50 mM	500 uL
DTT	0.5 mM	50 mM	500 uL
PMSF (in DMSO)	1.0 mM	100 mM	500 uL

How to make TEA buffer ???

• Add glass beads, to the mixture, equal to the amount of cells (usually 100uL - 200uL), use a pcr tube (200uL)
• Intercalate between ice freeze and vortex ~1min (in each) for about 10min.
• Centrifuge the cells at 5000g for 5min. at 4ºC
• Harvest the supernatant into a fresh 1.5mL Eppendorf and store it at -20ºC till further use.