SporulationinLiquidMedia

1)Growcellsin25mlsofPSP2(in250mlflask)supplementedwith25%oftherecommendedconcentrationofaminoacidsuntiltheyareabout1x10⁷cells/ml(~1day).2)Washwithoncewith25mlsofsterilewater.3)ResUSPendin25mlsofSPMmedia.4)Leave1to2daysandcheckundermicroscopefortheformationoftetrads.Totetraddissect:6)Transfer1mltoa1.5mlmicrofugetube.7)Spindownandsuckoffs/n.8)Resuspendin950µlofP-solution.9)Add50µlof10mg/mlzymolyase(besuretospindownthezymolyasebeforeuse).10)Letstand@roomtemp.for~10minsthenputonice.Checkasmallsampleunderthescopetoseeifthecellwallhasbeendigested.Try5moreminutesiftheyaren’tdigestedyet.11)Drip20µlontoaYEPDplateinalinenearthetopoftheplate.Thentilttheplatebackandforthuntilyourdripsruntogetherandyouhaveaniceline.Marktheedgesofyeastlineonthebottomoftheplatetohelplineupyourplateonthescope.*Idealtetraddissectingplatesshouldbepouredthinandeven,andstoredatroomtemperature.*Onceyouhavedrippedyouryeastontheplateitcanbeusefultolettheplatesitforacoupleofhourssothatthesporescansettleinandspreadout,butitisnotnecessary.

PSP2

SPM

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