Materials:
- RNase A: Make up as a solution in water at 10 mg/mL, Heat for 10 min in a boiling water bath or heating block to eliminate any DNase activity. Aliquot and store at -20oC.
- 0.4 M Ammonium acetate.
- Chloroform= A 24: 1 mix of chloroform and isoamyl alcohol. Store at 4oC.
- Phenol/chloroform= 25:24: 1 mix of TE-equilibrated phenol, chloroform, and isoamyl alcohol. Store at 4oC.
- 100% Ethanol.
- Sterile wooden toothpicks.
- Add 50 microliters of 4 M ammonium acetate containing 200 micrograms/mL RNase A to each miniprep and incubate it at room temperature for 20 min.
- Add 100 microliters of phenol/chloroform to each DNA preparation.
- Vortex briefly and centrifuge at high speed for 2 min in a microfuge. Remove the top layer containing the DNA and place it in a new sterile tube.
- Add 100 microliters of chloroform to each tube.
- Vortex briefly and centrifuge at high speed in a microfuge for 2 min. Remove the DNA in the top layer and place it in a second sterile tube.
For phenol/chloroform extractions avoid removing material from the interface.
- Add 200 microliters of 100% ethanol to each tube.
- Shake briefly to precipitate the DNA and centrifuge at high speed for 5 min at room temperature.
Reference: Number 18 on References