The extraction of nucleic acids involves adding an equal volume of phenol-chloroform to an
aqueous solution of lysed cells or homogenized tissue, mixing the two phases, and allowing
the phases to separate by centrifugation.

Centrifugation of the mixture yields two phases: the lower organic phase and the upper aqueous phase. Chloroform mixed with phenol is more efficient at denaturing proteins than either reagent is alone. The phenol-chloroform combination reduces the partitioning of poly(A)+ mRNA into the organic phase and reduces the formation of insoluble RNA-protein complexes at the interphase. Isoamyl alcohol is added to prevent foaming.

The aqueous phase is on top because it is less dense than the organic phase (phenol:chloroform). The proteins will partition into the lower organic phase while the nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in the upper aqueous phase. The upper aqueous phase is pipetted off and care is taken to avoid pipetting any of the organic phase or material at the interface. This procedure is often performed multiple times to increase the purity of the DNA.

The aqueous phase is then treated with monovalant salts with high acidic pH to raise the salt concentration and low pH gradient, and therefore cause the nucleic acid to precipitate out of solution.
Absolute alcohol is then added to precipitate out the DNA, which is then pellet down, washed and eluted for further use.