Isolating Vacuoles from yeast grown in -Ura minimal media

[Kevin Morano]

Morris F. Manolson (mm6y+ at andrew.cmu.edu) wrote:
: 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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Morrie,

In my lab (Klionsky), we use an adaptation of the Anraku method which 
works very well for us. This method involves growing the cells in 
Wickerham's minimal and making spheroplasts. The sphero's are lysed using 
DEAE-dextran, on ice, and the whole shebang is loaded at the bottom of an 
ultra tube and vacuoles floated up. This has worked fine with URA plasmids.

See the 1992 paper by Yaver, et al., I think it describes the protocol, 
or e-mail me for more info.
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Kevin A. Morano
Internet:kamorano at ucdavis.edu    
Bitnet:kamorano at ucdavis   
Section of Microbiology, University of California, Davis