Phenol extraction of DNA samples ?

Monday, September 22, 2003

Description
Phenol extraction is a common technique used to purify a DNA sample (1). Typically, an equal volume of TE-saturated phenol is added to an aqueous DNA sample in a microcentrifuge tube. The mixture is vigorously vortexed, and then centrifuged to enact phase separation. The upper, aqueous layer carefully is removed to a new tube, avoiding the phenol interface and then is subjected to two ether extractions to remove residual phenol. An equal volume of water-saturated ether is added to the tube, the mixture is vortexed, and the tube is centrifuged to allow phase separation. The upper, ether layer is removed and discarded,
including phenol droplets at the interface. After this extraction is repeated, the DNA is concentrated by ethanol precipitation.?br>
Primary Author
Bruce A. Roe ( broe@ou.edu )
?br>Affiliation
University of Oklahoma , United States
?br>Co-Author(s)
Judy S. Crabtree
Akbar S. Khan?

Procedure
Procedure
?1. Add an equal volume of TE-saturated phenol to the 1. DNA sample contained in a 1.5 ml microcentrifuge tube and vortex for 15-30 seconds.

2. Centrifuge the sample for 5 minutes at room temperature to separate the phases.

3. Remove about 90% of the upper, aqueous layer to a clean tube, carefully avoiding proteins at the aqueous:phenol interface. At this stage the aqueous phase can be extracted a second time with an equal volume of 1:1 TE-saturated phenol:chloroform, centrifuged and removed to a clean tube as above but this additional extraction usually is not necessary if care is taken during
the first phenol extraction.

4. Add an equal volume of water-saturated ether, vortex briefly, and centrifuge for 3 minutes at room temperature. Remove and discard the upper, ether layer, taking care to remove phenol droplets at the ether:aqueous interface. Repeat the ether
extraction.

5. Ethanol precipitate the DNA by adding 2.5-3 volumes of ethanol-acetate ?

Recipes
This is the recipe of my protocol

Supplies
This is the stuff I need for my protocol

Tips
Here are some pointers on how to work with my protocol

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Phenol extraction of DNA samples ?