Phenol:Chloroform Extraction and Ethanol Precipitation of DNA

[Luke]

Phenol:Chloroform Extraction and Ethanol Precipitation

Buffers:
•   3M Sodium Acetate pH 5.2
•   80% Ethanol
•   100% Ethanol
•   Phenol:Chloroform:Isoamyl Alcohol mixture


1.   Add 1 volume Phenol:Chloroform:Isoamyl Alcohol to tube with DNA (ie 100uL each)
2.   Vortex 30-60 seconds.
3.   Centrifuge 4-5 minutes at max speed (13,200 RPM)
4.   Remove aqueous (top) layer into new tube, do not take any of bottom layer.
5.   Back extract by adding 1 volume sterile water to phenol mixture, repeat steps 2-4.
6.   Add 1/10 volume 3M Sodium Acetate pH 5.2. mix thoroughly by pippetting
7.   Add 2.5 volumes ice cold 100% Ethanol, mix thoroughly
8.   Place in dry ice ethanol bath (-70) or -80 freezer for at least 30 minutes.
9.   Centrifuge at max speed at 4 C for 30 minutes.
10.   Remove supernatant avoiding DNA pellet (clear to whitish)
11.   Wash pellet with 80% ice cold ethanol
12.   Centrifuge at 4 C for at least 10 minutes
13.   Remove ethanol gently
14.   Air dry pellet or speed vac dry
15.   Add appropriate volume water, let sit without re-suspension on ice for several minutes
16.   Re-suspend pellet