Zymolyase protocol

fengyang fengyang at PRINCETON.EDU
Mon Sep 2 14:06:15 EST 1996

Dear All:

I use zymolyase to break down yeast cell wall and then do immunostaining.
For some reason the zymolyase reaction only gave 10-20% dark cells under microscope.
The conditions are following:
10^7 cells of overnight culture (OD596=1)
spin to remove media
wash 2x with 0.1M KPO4(pH6.5)/1.2M sorbitol
suspend cells in  0.5mL 0.1M KPO4(pH6.5)/1.2M sorbitol
add 5ul mecaptoethanol, 5ul 10mg.mL zymolyase in 0.1M KPO4(pH6.5)/1.2M sorbitol
30'/37degree shaking

Can somebody offer some advices/protocols?

Many thanks!


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