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Protocol C.3

Chromosomal DNA Preparation

This protocol was developed for cultured cells but should be appropriate for dissociated tissues as well.


HMW Lysis Buffer

10 mM Tris 7.5 1 ml 1M Tris 7.5

5 mM EDTA 8.0 1 ml 500 mM EDTA 8.0

0.4 M NaCl 8 ml 5M NaCl

up to 100 ml with Q

Other Reagents: Proteinase K (10 mg/ml in Q), DNase Free RNaseA (2 mg/ml), 20% SDS


• Pellet 30 ml of cultured cells at 1x106 cells/ml by spinning at 1.2K for 10 minutes.

• Wash with 10ml 1X PBS and resuspend in 4 ml HMW Lysis Buffer.

• Add 40 ml 20% SDS and 110 ml RNaseA. Tilt for at least 6 hours at 37°C.

• Add 90 ml Proteinase K and tilt for 4-6 hours at 37°C.

• Add 2 ml Phenol/Chloroform and mix by inversion. Spin at 3K for 10 minutes.

• Discard the gooey globs of chromosomal DNA/Protein and save the aqueous phase in another tube.

• Add 2 volumes of cold EtOH and invert 5-6 times.

• Pull out the stringy white precipitate with a glass hook (should not be gooey) and place in an eppendorf with 500 ml TE. Hold at 4°C, do not freeze. I usually get 1-2 mg/ml.