Quick and Easy Isolation of Genomic DNA from Yeast
Procedure
Transfer1.5mlofliquidcultureofyeastgrownfor20-24hat30°CinYPD(1%yeastextract,2%peptone,2%dextrose)intoamicrocentrifugetube.Pelletcellsbycentrifugationat20,000×gfor1-5minutes.Add200µlofHarju-bufferImmersetubesinadryice-ethanolbathfor2minutes,Transfertoina95°Cwaterbathfor1minute.RepeatthelasttwostepsVortex30seconds.Add200µlofchloroformandvortex2minutes.Centrifuge3minutesatroomtemperature,20,000×g.Transfertheupperaqueousphasetoamicrocentrifugetubecontaining400µlice-cold100%ethanol.Mixbyinversionorgentlevortexing.Incubateatroomtemperature,5minutes.Alternatively,precipitateDNAat-20°Ctoincreaseyield.Centrifuge5minutesatroomtemperature,20,000×g.RemovethesupernatantwithapulledPasteurPipettebyvacuumaspiration.Washthepelletwith0.5ml70%ethanolCentrifuge5minutesatroomtemperature,20,000×g.Removesupernatant.Air-drythepelletsatroomtemperatureorfor5minutesat60°Cinavacuumdryer.ResUSPendin25-50µlTE(pH8.0)]orwater.Samplesobtaineddirectlyfromplatesshouldberesuspendedina10µlvolume,becausetheyieldwillbesmaller.0.25µlRNasecocktailshouldbeaddedtothesamplesusedforSouthernblothybridization(finalconcentration0.125URNAseA,5URNaseT1).Reagents
Harju-Buffer–2%TritonX-100–1%SDS,–100mMNaCl–10mMTris-HCl,pH8.0,–1mMEDTA
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