Peccoud Lab Protocol: Isolating genomic DNA or plasmids from yeast

Introduction

The following protocol will recover total DNA and, with the indicated modifications, is useful to recover plasmids or genomic DNA. For plasmid recovery, the total DNA is transformed into E. coli. For genomic DNA to be used for PCR amplification – such as testing for gene deletions or fusions – one can skip the RNAse treatment. For extraction of genomic DNA for sequencing, there are additional steps for improving sample quality.

Materials

 

  1. lysis buffer
  • 2% Triton X-100
  • 1% SDS
  • 0.1 M NaCl
  • 1mM EDTA pH 8.0
  • 10mM Tris-HCl pH 8.0

To make 50 mL solution

  1. Glass Beads, Acid Washed 425-600 : 0.4-0.5 mm glass beads (sterilize in 100 mL bottle by autoclaving on wrapped cycle)
  2.  phenol:chloroform:isoamyl alcohol mixture, pH6.7/8.0  (25:24:1) (in fume hood)
  3. 1x TE
  • 10mM Tris-HCl pH 8
  • 1mM EDTA pH 8

To make 50mL solution

  1.  RNase A 20 mg/ml solution  free of DNase (in -30 °C freezer, drawer 4)
  2. Screw cap flat bottom microcentrifuge tubes
  3. 1.5 mL microcentrifuge tubes

 Method

  1. Place 50 mL tube of 95% ethanol, and 50 mL tube of 70% ethanol in -30C freezer before starting — DO NOT STORE THESE IN FREEZER
  2. Grow yeast cultures, fresh cultures are best
  3. Decant cells into screw cap tube and centrifuge at 14,000 rpm for 30 s. Aspirate media and repeat.
  4. Add 250 uL of lysis buffer and resuspend pellet
  5. Add acid washed glass beads until they are just covered by the lysis buffer.
  6. In fume hood, add 400 uL of phenol:cholorform:isoamyl alcohol
  7. Vortex at top speed for 2 minutes for plasmids and 5 minutes for genomic DNA (use bead-beater vortexer)
  8. Centrifuge for 5 min at 14,000 rpm and transfer aqueous layer (top) to new screw cap tube
  9. Add 600 uL of cold 95% ethanol and keep tube at -30 °C for 30 min
  10. Pellet DNA by centrifugation at 14,000 rpm at 4 °C for 15 min, and aspirate ethanol
  11. Dry pellet by air-drying (30 min) or vacuum drying

If isolating plasmids, pellet may be resuspended in 200 uL water and transformed into E. coli. If isolating genomic DNA for PCR, resuspend pellet in 400 uL water and use 1-2 uL in PCR reaction.

If isolating genomic DNA for sequencing, continue as follows:

  1. Resuspend dried pellet in 200 uL TE – let it sit for 15 min, vortex and briefly spind down, and let it sit for another 15 min
  2.  Add 5 uL of RNase A and incubate at 37 °C for 20 min
  3. Add 300 uL phenol:cholorform:isoamyl alcohol and vortex for 2 min
  4. Remove top, aqueous layer and place in new screw cap tube
  5. Add 300 uL of cholorform, vortex for 2 min
  6. Remove top, aqueous layer and place in new 1.5 mL microcentrifuge tube
  7. Add 8 uL of 5M NaCl and 2 volumes (400 uL) of cold 95% ethanol, place at -30 °C for 30 min
  8. Pellet DNA via centrifugation at 14,000 rpm at 4 °C for 15 min, and aspirate ethanol
  9. Wash with cold 70% ethanol, let sit for 10 min, aspirate ethanol, and dry
  10. Resuspend the pellet in water or TE