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spheroplasting problem

Lynn S Dahleen dahleen at badlands.NoDak.edu
Tue Aug 30 17:13:45 EST 1994


Unto The knowledgable of the World,
	I have been having problems spheroplasting my yeast for use in 
YAC transformations.  I have essentially been using the protocol 
described by Parcival et al, using lyticase (Fluka BioChemika).  The 
enzyme appears to work, but much slower than it should.  It usually takes 
45 min rather than the published 15 min. to reach 95% spheroplasting.  I 
have heard that if it takes longer than 15 min., something is wrong, and 
as I do not get any transformants (even with my + control) I tend to 
agree.  My questions are:  Could I try adding more lyticase (>1000 units/50 
mls of cells)? Should I try a different source for lyticase?  Should I 
alter my growing conditions to get younger cells (I normally use an 
overnight culture grown to the published optimal density?  Any help would 
be appreciated.  Thanks in advance!
Dave Horvath
USDA/ARS/NCSL




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