Phenol–chloroform extraction

Phenol–chloroform extraction is a liquid-liquid extraction technique in molecular biology used to separate nucleic acids from proteins and lipids. 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. Phenol only has a slightly higher density than water therefore it is mixed with chloroform to form a mixture with a much higher density than water. The aqueous phase is on top because it is less dense than the organic phase (phenol:chloroform). The hydrophobic lipids will partition into the lower organic phase, the proteins will remain at the interphase, 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. This procedure yields large double stranded DNA that can be used in PCR or RFLP. The disadvantages of this technique in forensic use, is that it's time-consuming, uses hazardous reagents and because it is a two-step process involving transfer of reagents between tubes it is at a greater risk from contamination.