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Genomic DNA Preps

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 YPAD at 30°C.
2. Collect the cells by centrifugation for 2 minutes 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 seconds in a microfuge.
3. Decant the supernant and briefly vortex the tube to resuspend the pellet in the residual liquid.  It is also possible to use cells grown on a Petri plate instead of growing cells in liquid culture.  In this case, collect cells from solid media with a sterile toothpick and suspend cells in lysis (Blue) buffer directly before adding phenol:chloroform.
4. Add 200 µl lysis mix (also referred to as Blue buffer). **work in hood when working with phenol** Add 200 µl phenol:chloroform:isoamyl alcohol (25:24:1). Add ~0.3 g of acid washed glass beads.
5. Vortex for at least 30 minutes at 4°C (cold room).
6. Add 200 µl TE (pH 7.5)
7. Centrifuge for 5 minutes at full power. Transfer aqueous layer to a fresh tube. Dispose phenol tube in phenol:chloroform waste. Add 750 µl of 95% ethanol. Mix by inversion. **no longer need to work in hood** Put in -20°C freezer for 1 hour.
8. Centrifuge for 5 minutes at full power. Discard the supernatant in the ethanol waste. Dry pellet completely by incubating at air temperature, at 37°C, or in the speed vac dryer.  Resuspend the pellet in 40 µl TE (pH 7.5).  Store prep at -20°C.
 
 
Lysis Mix (Blue Buffer)
200 µl Triton X-100
1 ml 10% SDS
200 µl 5M NaCl
100 µl 1M Tris pH 8
20 µl 0.5M EDTA
8.48 ml ddH2O
TOTAL 10 ml