Phenol extraction

Phenol (C₆H₅OH) is a water-soluble compound consisting of a phenyl group (-C₆H₅) bonded to a hydroxyl group (-OH).

A mixture of TE (or Tris-Ethylenediaminetetraacetic acid) and phenol is combined with an equal volume of an aqueous DNA and RNA sample. After agitation and centrifugal separation, the aqueous layer is extracted and further processed with ether and the DNA is concentrated by ethanol precipitation.

The phenol extraction technique is often used to purify samples of nucleic acids taken from cells.[1] To obtain nucleic acid samples, the cell must be lysed and the nucleic acids separated from all other cell materials. Phenol is a useful compound for breaking down superfluous cell materials that would otherwise contaminate the nucleic acid sample.

Phenol extraction of nucleic acids works as a result of its non-polar nature and its higher density than water (1.07 g/cm³[2] compared to water’s 1.00 g/cm³). This means in a water-phenol solution, denatured proteins and other cell components will be dissolved in the phenol, leaving nucleic acids dissolved in the water.[3] The solution can then be centrifuged to separate the phenol from the water into corresponding organic and aqueous phases. The nucleic acid containing aqueous phase can then be extracted.

Phenol is often used in combination with chloroform.[4] The purpose of adding chloroform along with phenol is to ensure a clear separation between the aqueous and organic phases. Chloroform and phenol are miscible, unlike phenol and water. The density of chloroform is 1.47 g/cm³, higher than that of water and phenol. Mixing chloroform and phenol creates a denser solution than phenol alone, therefore the separation of the organic from the aqueous phase is even clearer than if only phenol was added to a cell sample. There is less cross-contamination from the organic phase in the aqueous phase. This is useful when the aqueous phase is removed from the solution to obtain a pure nucleic acid sample.

For phenol to be effective, the pH of the solution must vary according to what is being extracted. In the case of DNA purification, a pH of 7.0–8.0 is used. If an experiment aims to obtain samples of purified RNA, a pH of around 4.5 is used. Due to the negative charge on the backbone of DNA from phosphates, decreasing the pH of a solution will lead to neutralization. A pH of 4.5 has a higher concentration of H+ ions that would neutralize the negative phosphate charges and cause DNA to dissolve in the organic phase, while RNA has an additional hydroxyl group in pentose sugar which allows the RNA to remain in the water phase.

• Phenol-chloroform extraction

• Physiology.med.cornell.edu
• Wisegeek.com