Yeast Spore Enrichment for Random Spore Analysis
(Ref.: Rockmill, B., E.J. Lambie, and G.S. Roeder.
1991. Spore enrichment. Methods Enzymol. 194:146-149.)
1. Prepare a suspension from Å1X108 cells and asci of a sporulated culture in
a polypropylene 1.5 ml µfuge tube containing 180 µl sterile DW. If sporulation
was carried out on KAc plates, then suspend cells scraped off of plate in
sterile DW. If sporulation was carried out in KAc broth, then wash cells and
asci 2X in 1 ml sterile DW before suspending in 180 µl DW.
2. Add 20 µl of a 5 mg/ml solution of
Zymolyase 20T in ZL buffer to the suspension and mix. Incubate at 30ûC for
between 20 and 60 min depending on how quickly the digestion progresses. Follow
the progress of the digestion microscopically.
ZL
Buffer For
100 ml
0.1
M NaPO4, pH 6.5 10
ml 1.0 M NaPO4, pH 6.5
1.2
M Sorbitol 21.9
g Sorbitol
40%
Glycerol 80
ml 50% Glycerol
DW Up
to 100 ml
Filter sterilize.
Dissolve 5 mg Zymolyase 20T in 1 ml ZL Buffer.
Store solution at -20ûC in a non-frost-free freezer.
3. Spin 30 sec in µfuge to pellet spores
and cells. Discard supernatant.
4. Suspend pellet in 1 ml DW and respin
in µfuge. Discard supernatant.
5. Suspend pellet in 0.1 ml DW and
vortex on high speed for 2 min.
6. Pour the liquid out of the µfuge tube
and rinse the tube 2X with 1 ml DW. Pour liquid out of tube each time. (Note:
The hydrophobic spores will stick to the µfuge tube wall while the cells and
debris will remain mostly in suspension.)
7. Add 1.0 ml sterile 0.01% NP-40 to the
tube and sonicate on ice for 2 min using the microtip of the Branson
Sonifier with the Output Control set at 1 and the % Duty Cycle set at constant.
(Note: This should give an Output level reading of Å20.)
8. Prepare 4X 1:10 serial dilutions of
the sonicated suspension (50 µl into 450 µl 0.01% NP-40).
9. Spread 200 µl of each dilution on a
YEPD plate or selective plate depending upon the phenotype of the desired
meiotic segregants.