- Spooled DNA
- SSC buffer
- Pancreatic ribonuclease A (100 µg/mL)
- Sodium lauryl sulfate (SLS)
- Sodium perchlorate
- Chloroform:isoamyl alcohol (24:1)
- 95% and 70% (v/v) ethanol
- Refrigerated centrifuge, rotor, and tubes
- 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
- Add pancreatic ribonuclease A to a final concentration of 100 micrograms/
mL and agitate slowly at 37°C for 1 hour.
- Add pronase to a final concentration of 50 micrograms/mL and again
agitate slowly at 37°C for another hour.
- 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.
- Extract the solution with chloroform:isoamyl alcohol (24:1 v/v) by adding
an equal volume and shaking vigorously for at least 15 minutes.
- Place the solution into appropriate centrifuge tubes and centrifuge for
5 minutes at 800 xg and 4°C.
- 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.
- Wash the spooled DNA twice with cold 70% ethanol and store for future