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Isolation of Genomic DNA

Adapted from Hoffman and Winston Gene 57: 267-272 [1987]

Safety Notes

Phenol is highly corrosive and can cause severe burns. Gloves, protective clothing, and safety glasses should be worn when handling it. All manipulations should be carried out in a chemical hood. Any areas of skin that come in contact with phenol should be rinsed with a large volume of water or polyethylene glycol 400 and washed with soap and water; ethanol should not be used.

Chloroform is irritating to the skin, eyes, mucus membranes, and upper respiratory tract. It should only be used in a chemical hood. Gloves and safety glasses should be worn. Chloroform is a carcinogen and may damage the liver and kidneys.

Methods

  1. Grow a 5-ml culture to saturation in YPD at 30oC.
  2. Collect the cells by centrifugation for 2 min. in a clinical centrifuge. Remove the supernatant and resuspend the cells in 0.5 ml distilled water. Transfer the cells to a 1.5-ml microfuge tube and collect them by centrifugation for 5 sec. in a microfuge.
  3. Decant the supernant and briefly vortex the tube to resuspend the pellet in the residual liquid.
  4. Add 0.2 ml lysis mix. **work in hood when working with phenol** Add 0.2ml phenol:cholorform:isoamyl alcohol (25:24:1). Add 0.3 g of acid washed glass beads.
  5. Vortex for a few seconds to check for leaks. Then, vortex for 30min. at 4oC (cold room).
  6. Add 0.2ml TE (pH 7.5)
  7. Centrifuge for 5 min at full power. Transfer aqueous layer to a fresh tube. Dispose phenol tube in phenol:chloroform waste. Add 1 ml of 100% ethanol. Mix by inversion. **no longer need to work in hood** Put in -20oC freezer for 1 hr.
  8. Centrifuge for 5 min. at full power. Discard the supernatant in the ethanol waste. Resuspend the pellet in 0.4 ml TE (pH 7.5) plus 3 µ l of a 10 mg/ml solution of RNase A. Incubate for 30 min. in a 37oC (shake every 10 min. to help pellet dissolve). Add 10 µ l of 4 M ammonium acetate plus 1 ml of 100% ethanol. Mix by inversion. Put in freezer for at least 30 min.
  9. Centrifuge for 5 min. at full speed. Discard supernatant in ethanol waste. Air-dry the pellet and resuspend in 50-100 µ l of TE (pH 7.5).

Lysis Mix

  • 1ml 20% Triton X-100
  • 1ml 10% SDS
  • 200ml 5M NaCl
  • 100ml 1M Tris pH 8
  • 20ml 0.5M EDTA
  • 7.7ml dd H2O
  • TOTAL 10ml