Isolating Vacuoles from yeast grown in -Ura minimal media

[Morris F. Manolson]

Dear Buddies,  I am trying to prepare vacuoles from yeast grown in
-Ura minimal media and having NO luck (Sniff).  I must isolate
vacuoles from a strain which contains a URA3 based plasmid, so 
I am growing up several liters of the plasmid bearing yeast in
-Ura minimal media ($$$$!) and doing vacuole preps as described by
Anraku (you know.... make spheroplast, osmotically lyse the spheroplast,
and then float the buoyant vacuoles up on a 12% ficoll gradient).  I have
no problem getting spheroplast, but when I do the low speed (2000g)
spin to get rid of the whole cells, mitochondria, nuclei etc etc....
EVERYTHING comes down....I end up with a nice clear supernatent
and nothing floats up in 12% ficoll.  

I have tried:

1) growing the cells to different cell titers
2) different amounts of Lyticase for spheroplasting

with no absolutely no success.  I need help! (No snickers from certain 
people on the 2nd, 5th or 7th floor of the mellon Inst., thank you....
oh, go ahead, laugh yourself silly).  Does anyone have any 
suggestions?  Thanks in advance, Morrie




  
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Morris F. Manolson                     Tel:  416-813-6662  (office)
Division of Cell Biology                     416-813-5594  (lab)
Hospital for Sick Children                   416-813-5028  (FAX)
88 Elm St., McMaster building        email:  Morrie at resunix.ri.sickkids.on.ca
Toronto, Ontario, Canada
M5G 1X8