facs help

Gina Alvino alvino at u.washington.edu
Mon Aug 14 18:58:12 EST 2000


Hello Yeast People!

I am working on a strain of S.cerevisiae with a deletion in a single
gene.  This deletion causes the cells to become enlarged with a slower
cell cycle as compared to the isogenic wildtype.  Facs analysis on these
cells is difficult to interpret because the peaks are broader and the
profiles are just plain ugly.  When the cells are challenged with
temperature or drug, the profiles are simply a mess.  I have tried lots of
ways to adjust the profiles when scanning but nothing helps.  So I'm
curious to try different protocols for preparing the cells for
scanning.  My protocol calls for fixation in EtOH at 4C overnight.  The
cell pellet is treated with RNase at 0.25mg/ml at 50C for 1hr.  Proteinase K
is added and incubation goes for another hour at 50C.  The DNA is then
stained with propidium iodide and the cells are stored at 4C until ready
to use.  I have scanned on the same day or days later with no change in
the profile.

Suggestions?

Any thoughts and/or comments would be appreciated.

Gina Alvino
University of Washington
Seattle, WA  USA



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