Spore sonication

Lori J Latus llatus at magnus.acs.ohio-state.edu
Mon Jun 13 15:11:43 EST 1994


Dear S. cerevisiae  people,
We are attempting a mass spore analysis to identify surviving spores carrying 
two selectable markers; one marker is genomic and the other marker is carried 
on a CEN vector library.  Our approach is based on a mass sporulation of our 
diploid strain of interest followed by a treatment that selectively kills 
remaining diploids.  This sporulation soup is then treated with glucouronidase 
(1:10 dilution of a crude glucouronidase extract, 15 minutes at 37oC) and 
sonicated (5 seconds at 50% output, using a Branson Sonifier 250).  The yeast 
are then plated in triplicate on different selective media.  I have only played
with this a bit but so far none of the yeast have survived the treatment; I do 
not even observe colonies on non-selective media.  I suspect that I am blowing 
the yeast to bits.  Does anybody have any advice, particularly on tetrad 
sonication protocols?  Thanks in advance for the help!
L.J. Latus
Dept. of Molecular Genetics, OSU
llatus at magnus.acs.ohio-state.edu



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