This is a cached page for the URL (http://axon.med.harvard.edu/~cepko/protocol/mike/C3.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Protocol.template

Protocol C.3

Chromosomal DNA Preparation

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

Solutions

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

Procedure

• 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.