Drain excess media as well as possible and weigh

Harvest Cells in 1 liter bottles (4 Krpm for 10 min.). Drain excess media as well as possible and weigh cells. Expected yield is 20-35 g cells. Anything less than 18g will give poor extracts. If cells are overgrown, lyticase will work poorly in spheroplasting cells.

Resuspend cell pellets in 35 ml 50 mM Tris 7.5, 30 mM DTT. Usually this can be done by gently shaking the centrifuge bottles. Leave cells in 1 liter bottles. Incubate at 30o for 15 min.

Pellet cells (4K rpm for 8 min.) and resuspend in 20 ml YPD/S. Add 15 ml 2M sorbitol. Add 15 ml recombinant lyticase. Incubate at 30o with occasional gentle mixing.