spheroplasting problem

Jonathan Swaffield jon_swaffield at ncsu.edu
Tue Apr 14 16:49:38 EST 1998

In article <6gvn32$r6d at net.bio.net>, alcacer at usa.net wrote:

> Hi! I am a stage student in Bourgogne 
> University. I have  been having problems spheroplasting 
> my 48 h. cultures of S.cerevisiae with zymolyase. 
> I know spheroplasting younger cultures  ( at exponential 
> phase )is  easier but I would like to achieve spheroplasting  
> with 48 h. cells. If anyone could  give me a hint of how 
> to do that  I would be immensely gratefull !! Thank you !!   
> ____________________________________________________________________
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Have you tried pre-incubating your cells in spheroplasting buffer
containing high concentrations of beta-mercaptoethanol or DTT before the
enzymic digestion. Reduces the S-S bonds in the cell wall and increases
the "efficiency" of the enzyme.  See unit 13:13 in Current Protocols in
Molecular Biology (the "Big Red Book").  This talks about 30 mM DTT and 15
min at room temp.  If needed I'm sure you could "incubate" longer.  Good

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