QuantitativeMatingAssay

GuthrieandFink,GuidetoYeastGeneticsandMolecularBIOLOGy,1991

1.Growacultureofthestrainwhosematingefficiencyistobedeterminedtoadensityofabout1E7cells/mlinYEPD.Prepareasimilarcultureofanappropriatematingtypetesterstrain(PT1{=a}orPT2{=alpha}).2.Mix2E6cellsoftheexperimentalstrainwith1E7cellsofthetesterstrainandcollectthecellsona0.45祄pore,25mmnitrocellulosefilterdisk.Inaddition,collectcellsofeachstrainonseparatefilters(theseserveascontrolsforreversionand/orcontamination).3.PlacethefiltersonthesurfaceofaYEPDplate(cellsideup!)andincubateat30degCfor5hr.4.ResUSPendthecellsoneachfilterin1mlofY-minbroth,sonicatefor5-10sectodisruptclumps,anddiluteinY-minbroth.PlatethecellsfromthematingmixonY-minmediumtotiterdiploids.Likewise,platecellsfromthesinglestrainfiltersonSDmediumtocheckforreversionand/orcontamination.CalculatethematingefficiencybydeterminingthetiterofdiploidsplusthecellswhosematingefficiencyisbeingdeterminedbyplatingthecellsfromthematingmixfilteronY-minsupplementedwiththeauxotrophicMarkersoftheexperimentalstrain.Thematingefficiencyisexpressedasthetiterofdiploidsdividedbythetiterofdiploidsplusthetiteroftheexperimentalstrain.

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