DNA extraction from cultured cells

Overview

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.