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
