Purification of DNA

• Spooled DNA
• SSC buffer
• Pancreatic ribonuclease A (100 µg/mL)
• Pronase
• Sodium lauryl sulfate (SLS)
• Sodium perchlorate
• Chloroform:isoamyl alcohol (24:1)
• 95% and 70% (v/v) ethanol
• Refrigerated centrifuge, rotor, and tubes

1. Decant off the alcohol, and dissolve the extracted DNA in 30 mL of diluted SSC buffer (0.1 X SSC) in a 125-mL erlenmeyer flask. This will require some time, as polymerized DNA dissolves slowly. Gentle swirling of the material will help.
2. Add pancreatic ribonuclease A to a final concentration of 100 micrograms/ mL and agitate slowly at 37°C for 1 hour.
3. Add pronase to a final concentration of 50 micrograms/mL and again agitate slowly at 37°C for another hour.
4. Add sodium lauryl sulfate (SLS) to make a 1% concentration (w/v) and sodium perchlorate to a final concentration of 1 M. Agitate for 30 minutes at room temperature.
5. Extract the solution with chloroform:isoamyl alcohol (24:1 v/v) by adding an equal volume and shaking vigorously for at least 15 minutes.
6. Place the solution into appropriate centrifuge tubes and centrifuge for 5 minutes at 800 xg and 4°C.
7. Remove the upper aqueous phase, add 2 to 3 volumes of 95% cold ethanol, and respool the DNA from this solution onto a glass rod.
8. Wash the spooled DNA twice with cold 70% ethanol and store for future analysis.