( Cell Preparation Cells w...)Electrophoretic Karyotypes of Wine Strains of Saccharomyces cerevisiae

Cell Preparation
Cells were prepared by the agarose bead encapsulation method¹¹ with minor modifications. A 200 ml YEPD culture was grown at 30 C to an O.D.₆₀₀ of 1.0 and centrifuged at 400500 x g for 5 minutes at 4 C. Following removal of the supernatant, cells were resuspended in 10 ml of SE (75 mM NaCl, 25 mM Na₂ EDTA, pH 8.0), recentrifuged, washed twice, and resuspended in 4 ml SE. In a 45C water bath, cells were mixed with 5 ml 1% low melt agarose (Bio-Rad Laboratories) in SE, prior to addition of 20 ml temperature- equilibrated mineral oil (Squibb). The resultant emulsion was mixed vigorously for 30 seconds before it was quickly poured into a beakerkept in an ice bathcontaining 100 ml of ice-cold SE and a stir bar stirring at medium speed. The mixture was stirred for several minutes before being transferred to several graduated polypropylene tubes which were centrifuged as before at room temperature. The mineral oil was removed and any beads not pelleted were dispersed by repeated pipeting with a large-bore pipette.

After recentrifugation and removal of excess SE and beads that did not pellet, the remaining pellets were combined into a single tube and centrifuged. The supernatant was removed, the inside of the tube wiped with a tissue to remove excess mineral oil, the volume brought to 20 ml with 25 mM Na₂EDTA and the final SDS concentration to 1%. After repeated pipeting with a wide-bore pipette to break up clumps, the tube was taped to a platform shaker and shaken for 10 minutes at room temperature. Following recentrifugation and removal of the supernatant, 0.5 ml of 2-mercaptoethanol and 10 mg of zymolyase-100T (Seikagaku Kogyo Co.) were added, the v
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