Quick and Easy Isolation of Genomic DNA from Yeast
来自 : 蚂蚁淘
Procedure
- Transfer1.5mlofliquidcultureofyeastgrownfor20-24hat30°CinYPD(1%yeastextract,2%peptone,2%dextrose)intoamicrocentrifugetube.Pelletcellsbycentrifugationat20,000×gfor1-5minutes.
- Add200µlofHarju-buffer
- Immersetubesinadryice-ethanolbathfor2minutes,
- Transfertoina95°Cwaterbathfor1minute.
- Repeatthelasttwosteps
- Vortex30seconds.
- Add200µlofchloroformandvortex2minutes.
- Centrifuge3minutesatroomtemperature,20,000×g.
- Transfertheupperaqueousphasetoamicrocentrifugetubecontaining400µlice-cold100%ethanol.Mixbyinversionorgentlevortexing.
- Incubateatroomtemperature,5minutes.Alternatively,precipitateDNAat-20°Ctoincreaseyield.
- Centrifuge5minutesatroomtemperature,20,000×g.
- RemovethesupernatantwithapulledPasteurPipettebyvacuumaspiration.
- Washthepelletwith0.5ml70%ethanol
- Centrifuge5minutesatroomtemperature,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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