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| Sporulation | ||
| Yeast sporulation (liquid) 1. Grow a saturated yeast culture in YEPD. 2. Take 0.2 ml and put into 5 ml sterile water. Spin 3-5'. 3. Pour off water; resuspend cells in 5 ml 0.3% KOAc + 20 µg/ml Adenine + any required amino acids because of homozygous mutations. 4. Incubate with aeration for 3-5 days at 23ºC (usually 4). Observe by microscopy-you are looking for nicely formed tetrads. 5. Cells can be stored at 4ºC. Dissection: 1. Wash 0.2 ml cells in 5 ml sterile water; spin as before. 2. Resuspend cell pellet in 0.25 ml sterile water. 3. Add 10 µl glusulase and incubate 10 minutes at room temp, then several hours on ice (overnight OK). 4. Spread 50 µl on a YEPD plate (top third of the plate), let dry and dissect. 5. TIP: inspect the plate-it should be dry, even and rather thick. 6. See Cora Style's web site for more tips http://www.tiac.net/users/cstyles/Needle.htm also http://www.tiac.net/users/cstyles/Idios.htm |
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| Comments: Some amino acids added as supplements can act as a nitrogen and carbon source and thereby inhibit sporulation like histidine. Sporulation can in some cases be achieved by lower amounts or leaving them out all together (stored pools from mitosis are enough). | ||
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