Phenol Extraction of DNA
Protocol 1
[edit]1. Add an equal volume of phenol to the DNA solution and vortex gently.
2. Separate the aqueous phase (top) which contains the DNA from the organic phase (bottom) by centrifugation in the microfuge, at 13000 rpm for 1 min.
3. Transfer the aqueous phase with care into a fresh microfuge tube and add an equal amount of 24:1 (v/v) chloroform-isoamyl alcohol. (isoamyl alcohol is Isopentyl alcohol, Isopentanol, Isobutylcarbinol)
4. In order to precipitate the DNA, add a 0.1 volume of 3 M sodium acetate, pH 5.5, to the aqueous phase and then 2 volumes of absolute ethanol or 1 volumn of isopropanol. Put into dry ice for 10 min.
5. Recover the precipitated DNA by centrifugation in the microfuge at 13000 rpm for 10 min.
6. Wash pellet with 75% EtOH once, spin for 3 min at 13000 rpm.
7. Dry DNA in biosafety hood.
8. Dissolve dried DNA in sterile TE or EB, pH 8.0, or water, and stored at 4 °C (or -20 oC).
Protocol 2
[edit]Extraction with Phenol:Chloroform:Isoamyl Alcohol
1. After digestion, add 70 ÎŒl 1X TE Buffer (pH 8.0) and 100 ÎŒl phenol:chloroform:isoamyl alcohol (25:24:1).
2. Vortex thoroughly.
3. Spin the tube in a microcentrifuge at 14,000 rpm for 5 min at 4°C to separate phases.
4. Carefully transfer the top aqueous layer to a clean 1.5-ml microcentrifuge tube. Discard the interface and lower phase into an organic waste container.
5. Add 400 ÎŒl 95% ethanol, 25 ÎŒl 10 M NH4OAc (or 1/10 volume of 3 M NaOAc), and 1 ÎŒl glycogen (20 mg/ml).
6. Vortex thoroughly.
7. Spin the tube in a microcentrifuge at 14,000 rpm for 5 min at 4°C.
8. Remove and discard the supernatant.
9. Carefully overlay the pellet with 300 ÎŒl 70% ethanol.
10. Spin in a microcentrifuge at 14,000 rpm for 2 min at room temperature.
11. Carefully aspirate off the supernatant.
12. Air dry the pellet for approximately 15 min at room temperature to evaporate residual ethanol.
13. When the pellet is dry, dissolve the DNA precipitate in 10 ÎŒl sterile 1X TE Buffer (pH 8.0) and store at â20°C until use.