Phenol–chloroform extraction is a liquid-liquid extraction technique in

molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...

used to separate nucleic acids from

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...

s and

lipids Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...

Process

Aqueous samples, lysed cells, or homogenised tissue are mixed with equal volumes of a

phenol Phenol (also called carbolic acid) is an aromatic organic compound with the molecular formula . It is a white crystalline solid that is volatile. The molecule consists of a phenyl group () bonded to a hydroxy group (). Mildly acidic, it ...

: chloroform mixture. This mixture is then centrifuged. Because the

: chloroform mixture is immiscible with water, the centrifuge will cause two distinct phases to form: an upper aqueous phase, and a lower organic phase. The aqueous phase rises to the top because it is less dense than the organic phase containing the phenol:chloroform. This difference in density is why phenol, which only has a slightly higher density than water, must be mixed with chloroform to form a mixture with a much higher density than water. The hydrophobic lipids will partition into the lower organic phase, and the proteins will remain at the interphase between the two phases, while the nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in the upper aqueous phase. The upper aqueous phase can then be pipetted off. Care must be taken to avoid pipetting any of the organic phase or material at the

interface Interface or interfacing may refer to: Academic journals * ''Interface'' (journal), by the Electrochemical Society * '' Interface, Journal of Applied Linguistics'', now merged with ''ITL International Journal of Applied Linguistics'' * '' Int ...

. This procedure is often performed multiple times to increase the purity of the DNA. This procedure yields large double stranded DNA that can be used in PCR or RFLP. If the mixture is acidic, DNA will precipitate into the organic phase while RNA remains in the aqueous phase. This is because DNA is more readily neutralized than RNA. There are some disadvantages of this technique in forensic use. It is time-consuming and uses hazardous reagents. Also, because it is a two-step process involving transfer of reagents between tubes, it is at a greater risk of

contamination Contamination is the presence of a constituent, impurity, or some other undesirable element that spoils, corrupts, infects, makes unfit, or makes inferior a material, physical body, natural environment, workplace, etc. Types of contamination ...

References

Molecular biology Biochemistry methods {{molecular-biology-stub

Process

See also

References