Protocol: 1.PatchcellsthathavebeenfreshlygrownonYPDtofreshlypreparedGNApre-sporulationplatesfor1dayat30°C.RepatchtoanotherfreshlypreparedGNApre-sporulationplateforonemoredayat30°C. 2.TransfertoliquidsporulationmediumbyresUSPendingasmallamountofcellsint2mlsofsupplementedliquidsporulationmedium.Don"tputintoomanycells! 3.Incubateonarollerwheelfor5daysat25°Cfollowedby3daysat30°C.Thesporulatedculturescanbestoredat4°Cforuptooneweekbeforedissection;olderculturesloseviABIlity. Sporulationefficienciesshouldbearound30%. MaterialsandReagents: GNAPresporualtionPlates Autoclavefor15minutes.Platesshouldalwaysbefreshlymadeandnomorethan1-2daysold. SupplementedLiquidSporulationMedium 2mls ThisisanupdatedsporulationfromLindaRilesandMattCurtiss.AlternateSporulationMethodforBY4743
FinalConcentrations recipefor2plates recipefor10plates 5%D-glucose 3%Difconutrientbroth 1%Difcoyeastextract 2%Bactoagar FinalConcentrations Stocksolutions 1%PotassiumAcetate 10%PotassiumAcetate(20g/200ml) 0.005%ZincAcetate 0.5%Zincacetate(1g/200ml) 1xurasupplement 100xurasupplementsolution 1xhissupplement 200xhissupplementsolution 1xleusupplement 200xleusupplementsolution
