Editing Phenol Extraction of DNA (section)

== Protocol 2<br>  ==

Extraction with Phenol:Chloroform:Isoamyl Alcohol<br>1. After digestion, add 70 ÎŒl 1X TE Buffer (pH 8.0) and 100 ÎŒl phenol:chloroform:isoamyl alcohol (25:24:1).

<br>2. Vortex thoroughly.

<br>3. Spin the tube in a microcentrifuge at 14,000 rpm for 5 min at 4°C to separate phases.

<br>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.

<br>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).

<br>6. Vortex thoroughly.

<br>7. Spin the tube in a microcentrifuge at 14,000 rpm for 5 min at 4°C.

<br>8. Remove and discard the supernatant.

<br>9. Carefully overlay the pellet with 300 ÎŒl 70% ethanol.

<br>10. Spin in a microcentrifuge at 14,000 rpm for 2 min at room temperature.

<br>11. Carefully aspirate off the supernatant.

<br>12. Air dry the pellet for approximately 15 min at room temperature to evaporate residual ethanol.

<br>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.<br>

[[Category:Protocols]]