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Quick and Easy Isolation of Genomic DNA from Yeast
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Procedure

  1. Transfer1.5mlofliquidcultureofyeastgrownfor20-24hat30°CinYPD(1%yeastextract,2%peptone,2%dextrose)intoamicrocentrifugetube.Pelletcellsbycentrifugationat20,000×gfor1-5minutes.
  2. Add200µlofHarju-buffer
  3. Immersetubesinadryice-ethanolbathfor2minutes,
  4. Transfertoina95°Cwaterbathfor1minute.
  5. Repeatthelasttwosteps
  6. Vortex30seconds.
  7. Add200µlofchloroformandvortex2minutes.
  8. Centrifuge3minutesatroomtemperature,20,000×g.
  9. Transfertheupperaqueousphasetoamicrocentrifugetubecontaining400µlice-cold100%ethanol.Mixbyinversionorgentlevortexing.
  10. Incubateatroomtemperature,5minutes.Alternatively,precipitateDNAat-20°Ctoincreaseyield.
  11. Centrifuge5minutesatroomtemperature,20,000×g.
  12. RemovethesupernatantwithapulledPasteurPipettebyvacuumaspiration.
  13. Washthepelletwith0.5ml70%ethanol
  14. Centrifuge5minutesatroomtemperature,20,000×g.
  15. Removesupernatant.
  16. Air-drythepelletsatroomtemperatureorfor5minutesat60°Cinavacuumdryer.
  17. 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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