Generally we are using the Qiagen DNeasy kit to extract DNA from cell pellets. Follow the instructions provided with the kit. The old-fashioned way is as follows.
Protocol
Adherent cells must be trypsinized first.
Centrifuge cells at 1500 rpm for 5 minutes. Pour off supernatant
Wash once in PBS
Wash once in STE
Resuspend in 1 to 3 ml of lysis buffer (depending upon size of homogenizer
Cell lysis buffer
Place tissue into Dounce homogenizer along with appropriate volume of lysis buffer, depending on size of homogenizer.
Grind up and down repeatedly.
Incubate 55°C in shaker at 250 rpm for 1 hr to overnight
Add 7.5 µl, 50 µl or 100 µl RNase A (2.0 mg/ml) to each sample and incubate 1 hr 37°C
Add equal volume of phenol, vortex and cetrifuge at 2000 rpm for 10 minutes.
Transfer aqueous (top) phase to clean tube
Add equal volume of 1:1 phenol/chloroform solution. Note: Use Glass pipette, not plastic.
Vortex and centrifuge at 2000 rpm for 10 minutes. Repeat until clean (1 or 2 times).
Add equal volume chloroform, vortex and centrifuge at 2000 rpm for 10 minutes. Note: Use Glass pipette, not plastic.
Transfer aqueous phase to a clean tube.
Add 2 volumes of -20°C 100% ethanol and place in -20°C freezer overnight.
Centrifuge 14,000 g for 15 min at 4ªC. Pour off supernatant
Let DNA pellet dry completely and resuspend in 100-200 µl TE.