Phenol–chloroform extraction

Phenol–chloroform extraction is a liquid-liquid extraction technique in molecular biology used to separate nucleic acids from proteins and lipids.

Aqueous samples, lysed cells, or homogenised tissue are mixed with equal volumes of a phenol:chloroform mixture. After mixing, the mixture is centrifuged and two distinct phases are formed, because the phenol:chloroform mixture is immiscible with water. The aqueous phase is on top because it is less dense than the organic phase (phenol:chloroform). The proteins and hydrophobic lipids 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.

If the mixture is acidic, DNA will precipitate into the organic phase while RNA remains in the aqueous phase due to DNA being more readily neutralised than RNA.

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