The phenol-chloroform method for nucleic acid isolation

Nucleic acids from cells can be isolated using the phenol-chloroform method. Phenol-chloroform is a mixture of buffer-saturated phenol and chloroform in 1:1 ratio. The steps in the procedure as as follows:

• 1) Lysis and homogenization in aqueous solution. This is done with a lysis buffer and by vortexing the sample.
• 2) Add phenol-chloroform to the lysate and mix it by vortexing it.

• 3) Centrifuge to separate the phases according to the density. Phenol has a density of 1.07 g/cm³ while chloroform has a higher density (1.47 g/cm³), thus they form two phases, the lower organic phase (phenol-chloroform) and the upper aqueous phase (see image below). Nucleic acids gain polarity from negatively charge phosphate backbone; therefore, nucleic acids are soluble in the upper aqueous phase. Proteins contain hydrophobic regions that interact with phenol and cause the proteins to precipitate at the interface between two phases (often as white flocculation). Lipids also have a hydrophobic region and will dissolve in the lower organic phase. The pH of the mixture determines the separation of nucleic acids. In neutral pH, RNA and DNA are retained in the upper aqueous phase, but in acidic pH, DNA moves to the lower organic phase and RNA remains in the upper aqueous phase.

• 4) Nucleic acid recovery. In the last step, nucleic acids are recovered from the aqueous phase by precipitation using isopropanol.

The phenol-chloroform extraction method is used for nucleic acid isolation.