Sporulation in Liquid Media
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Sporulation in Liquid Media
1) Grow cells in 25mls of PSP2 (in 250ml flask) supplemented with 25% of the recommended concentration of amino acids until they are about 1x107 cells/ml (~1 day).
2) Wash with once with 25mls of sterile water.
3) Resuspend in 25mls of SPM media.
4) Leave 1 to 2 days and check under microscope for the formation of tetrads.
To tetrad dissect:
6) Transfer 1ml to a 1.5ml microfuge tube.
7) Spin down and suck off s/n.
8) Resuspend in 950µl of P-solution.
9) Add 50µl of 10mg/ml zymolyase (be sure to spin down the zymolyase before use).
10) Let stand @ room temp. for ~10 mins then put on ice. Check a small sample under the scope to see if the cell wall has been digested. Try 5 more minutes if they aren’ t digested yet.
11) Drip 20µl onto a YEPD plate in a line near the top of the plate. Then tilt the plate back and forth until your drips run together and you have a nice line. Mark the edges of yeast line on the bottom of the plate to help line up your plate on the scope.
~undefinedIdeal tetrad dissecting plates should be poured thin and even, and stored at room temperature.
~undefinedOnce you have dripped your yeast on the plate it can be useful to let the plate sit for a couple of hours so that the spores can settle in and spread out, but it is not necessary.
PSP2
- 6.7g YNB without amino acids
- 1g yeast extract
- 10g potassium acetate
- Mix in 1L of 50mM potassium phthalate buffer (pH 5.0)
SPM
- 3.0g potassium acetate
- 0.2g raffinose
- Mix in 1L distilled water
