PHENOL EXTRACTION AND ALCOHOL PRECIPITATION

A. PHENOL EXTRACTION

Used to remove proteins, excess dNTPÕs, and other impurities in a nucleic acid prep.

1. Bring volume to at least 100 ul (100 ~ 200 ul) with dH₂O, TE, or DSB.

2. Add slightly less than equal volume of buffer-saturated phenol or phe:chl:IAA (90-190 ul). Invert tube and thoroughly mix layers.

Optional – incubate at 55-65' C for 10 minutes. Don't let phenol vapors build up pressure and pop cap – vent before incubation.

3. Spin in microfuge 2-5 min to separate phases. AQUEOUS layer on TOP, phenol layer on the bottom.

4. Carefully separate aqueous and phenol layers; draw off aqueous (top) layer into a separate tube. Use a small tip. OR suck up the phenol layer and dispose into organic waste. MOST important point is to be sure none of the organic phenol or "junk" remains in the aqueous sample.

5. Do a second extraction using chloroform. Add half the aqueous volume of chloroform, mix layers, microfuge to separate layers as before.

6. Carefully separate aqueous and chloroform layers. Dispose of chloroform in organic waste container.

B. ALCOHOL PRECIPITATION

Used to remove excess salts, change buffer, or complete the phenol extraction.

1. Add cold acetate salt to aqueous sample and mix:

a) for 3 M NaAc pH 4.8, add 10% of the total liquid volume (the aqueous plus the acetate salt; example for 100 ul aqueous add 11 ul acetate).

b) for 7.5 M NH₄Ac, add ½ of your aqueous volume.

2. Add cold alcohol and mix:

a) for 100% EtOH, add 2 times the total liquid volume (aqueous plus acetate salt).

b) for isopropanol, add 1.1x the total liquid volume (aqueous plus acetate salt). Use isopropanol if the DNA is in TBE buffer.

3. Put sample on dry ice (20-30 min), at -80' C or at -20' C (1-2 hours or ON).

4. Microfuge 15-20 minutes at full speed. Balance carefully.

5. Remove supernatant. You can draw it off, aspirate, or pour off. Be careful, do not dislodge your pellet!

6. Do an alcohol wash; add drops of cold 70% alcohol (200-400 ul) over pellet to dissolve salts.

7. Mix or vortex pellet, then spin in microfuge 15-20 minute to secure pellet. Carefully remove supernatant but do not dislodge the pellet.

8. Repeat 70% EtOH wash if needed.

9. Dry the pellet thoroughly since alcohol can inhibit reactions:

a) put in 37' C incubator (open the cap)

b) speedvac 5-15 minutes
c) use "DNA dryer" across the top of the open tube. Don't blow out your pellet.

10. Resuspend DNA thoroughly in appropriate volume of dH₂O, TE, or DSB. Pipette up and down the sides of the tube.