Phenol extraction
Phenol extraction is a processing technology used to prepare phenols as raw materials, compounds or additives for industrial wood processing and for chemical industries. Phenol extraction also is a laboratory process to purify DNA samples.
In this method, a mixture of TE (or Tris-Ethylenediaminetetraacetic acid) and phenol is combined with an equal volume of an aqueous DNA sample. After agitation and centrifugal separation, the aqueous layer is extracted, and further processed with ether. Then the DNA is concentrated by ethanol precipitation.
The phenol extraction technique is often used to purify samples of nucleic acids taken from cells. 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.
There are two reasons why phenol makes such an effective purifier for nucleic acid samples. The first is that it is a non-polar compound. Because nucleic acids are highly polar, they do not dissolve in the presence of phenol. The second is that phenol has a density of 1.07 g/cm3, which is higher than the density of water (1.00 g/cm3). Thus, when phenol is added to a cell sample solution the water and phenol remain separate. Two “phases” form when phenol is added to the solution and centrifuged. There is an aqueous, polar phase at the top of the solution containing nucleic acids and water, and an organic phase containing denatured proteins and other cell components at the bottom of the solution. The aqueous phase is always on top of the organic because, as mentioned above, phenol is denser than water. Nucleic acids are polar, and therefore stay in the aqueous phase, whereas non-polar cellular components move into the organic phase.[1][2] After phenol has been added to the sample it is centrifuged and the aqueous and organic (“Phenol”) phases form.
Phenol is often used in combination with chloroform. The purpose of adding chloroform along with phenol is to ensure a clear separation between the aqueous and organic phases. Chloroform and phenol mix well together, 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, and 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 for when the aqueous phase is removed from the solution in order to obtain a pure nucleic acid sample.
pH is an important factor to consider in the phenol extraction technique. 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 the aim of an experiment is to obtain samples of purified RNA, a pH of around 4.5 is used. Because of 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 additional hydroxyl group in pentose sugar which allows the RNA to remain in water phase.
See also
References
- ↑
- ↑ Oswald, Nick. "The Basics: How Phenol Extraction Works". Bitesize Bio.
