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Quick and Easy Isolation of Genomic DNA from Yeast

Author: Markus Ralser
Source: Protocol Online
Date Added: Mon Feb 02 2009
Date Modified: Mon Feb 02 2009
Abstract: This protocol describes a quick and easy method for genomic DNA preparation from yeast. The protocol can be used for PCR or southern blot analysis

Procedure

  1. Transfer 1.5 ml of liquid culture of yeast grown for 20 - 24 h at 30C in YPD (1% yeast extract, 2% peptone, 2% dextrose) into a microcentrifuge tube. Pellet cells by centrifugation at 20,000 g for 1-5 minutes.
  2. Add 200 l of Harju- buffer
  3. Immerse tubes in a dry ice-ethanol bath for 2 minutes,
  4. Transfer to in a 95C water bath for 1 minute.
  5. Repeat the last two steps
  6. Vortex 30 seconds.
  7. Add 200 l of chloroform and vortex 2 minutes.
  8. Centrifuge 3 minutes at room temperature, 20,000 g.
  9. Transfer the upper aqueous phase to a microcentrifuge tube containing 400 l ice-cold 100% ethanol. Mix by inversion or gentle vortexing.
  10. Incubate at room temperature, 5 minutes. Alternatively, precipitate DNA at -20C to increase yield.
  11. Centrifuge 5 minutes at room temperature, 20,000 g.
  12. Remove the supernatant with a pulled Pasteur pipette by vacuum aspiration.
  13. Wash the pellet with 0.5 ml 70% ethanol
  14. Centrifuge 5 minutes at room temperature, 20,000 g.
  15. Remove supernatant.
  16. Air-dry the pellets at room temperature or for 5 minutes at 60C in a vacuum dryer.
  17. Resuspend in 25- 50 l TE (pH 8.0)] or water. Samples obtained directly from plates should be resuspended in a 10 l volume, because the yield will be smaller. 0.25 l RNase cocktail should be added to the samples used for Southern blot hybridization (final concentration 0.125 U RNAse A, 5 U RNase T1).

Reagents

Harju- Buffer
2% Triton X-100
1% SDS,
100 mM NaCl
10 mM Tris-HCl, pH 8.0,
1 mM EDTA

Reference

Harju S, Fedosyuk H, Peterson KR. Rapid isolation of yeast genomic DNA: Bust n' Grab. BMC Biotechnol. 2004 Apr 21;4:8.; PMID: 15102338

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