Editing Phenol Extraction of DNA

== Protocol 1<br> ==

1. Add an equal volume of phenol to the DNA solution and vortex '''gently.''' 

<br>2. Separate the&nbsp; aqueous phase (top) which contains the DNA from the organic phase (bottom) by centrifugation in the microfuge,&nbsp; at 13000 rpm for 1 min. 

<br>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.&nbsp;(isoamyl alcohol is Isopentyl alcohol, Isopentanol, Isobutylcarbinol)''' 

<br>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&nbsp; into dry ice for 10 min. 

<br> 

5. Recover the precipitated DNA by centrifugation in the microfuge at 13000 rpm for 10 min. 

<br> 

6. Wash pellet with '''75% EtOH''' once, spin for 3 min at 13000 rpm. 

<br> 

7. Dry DNA in biosafety hood. 

<br> 

8. Dissolve dried DNA in sterile TE or EB, pH 8.0, or water, and stored at 4 °C (or -20 oC).<br> 

== 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]]