The Protein Man's Blog | A Discussion of Protein Research

While phenol extraction is one of the commonly used methods in purifying DNA samples (i.e., removing proteins from cell lysates during DNA preparation), some people do not fully understand how and why it works. Read on to learn and appreciate how phenol extraction works.

The Basic Protocol of Phenol Extraction

To fully understand how this method works, let us take a look at how the procedure is performed in the lab. Here’s a quick rundown of the basic protocol involved in phenol extraction.

1. Add an equal volume of TE-equilibrated (Tris-ethylenediaminetetraacetic acid) phenol to the aqueous DNA sample. Water and phenol have different polarities so they won’t mix. Instead, you’ll see two liquid phases. Phenol is denser than water (1.07 g/cm³ 1.0 g/cm³) so it will sit at the bottom while the water will stay on top.
2. Mix the phases to force the phenol into the water layer. An emulsion of droplets will form throughout the solution, and the proteins in the water phase will be denatured and eventually partition into the phenol. However, since the DNA’s phosphate backbone carries a negative charge, it will not dissolve in the phenol (a non-polar solvent) but will remain in the water phase (a polar solvent). On the other hand, the addition of phenol changes the solubility of proteins by forcing the hydrophobic non-polar residues (which usually hides on the inside of the proteins and away from the solvent) to interact with the solvent. Hence, the proteins are made more soluble in phenol than in water.
3. Vortex and spin the solution in a microcentrifuge for approximately 2 minutes to separate the phases.
4. Transfer the water phase (the upper layer) by carefully pipetting it to a new tube. Avoid pipetting the interface since it contains much of the stuff you want to discard (i.e., DNA molecules, partially denatured proteins, and partially denatured DNA binding proteins). If you think the volume of the aqueous phase is too low, simply add some TE buffer or double-distilled water (ddH₂0) and repeat the vortex and spin steps. Discard the protein/phenol solution.
5. Desalt and concentrate the DNA using ethanol precipitation.

Important Reminders:

• Always take the necessary precautions when working with phenol. Phenol is highly corrosive and may cause severe burns if not handled properly. To avoid any accidents, wear protective clothing (gloves, goggles, lab coat) and work in the fume hood when handling large quantities of phenol. Wash the affected area with water if you happen to spill some on yourself and seek immediate medical attention if the burns cause extensive damage.
• Avoid using old phenol since it can compromise the integrity of your DNA sample.
• Store phenol in the dark at approximately 4°C under the right conditions, phenol can be stored safely it for up to 3 months. When preparing your own phenol, it should be kept at -20°C. It is also important to remove the aliquot every 3 months.
• If you want to achieve a clearer separation between the phases and prevent phase inversion (the aqueous layer is under the phenol phase), add chloroform to buffer-saturated phenol. This will create a denser solution (the density of chloroform is 1.47 g/cm³) compared to using phenol alone and reduce the formation of a fuzzy interface. Additionally, it will lessen the risk of cross-contamination from the organic phase in the aqueous phase thereby increasing the purity of your yield.
• Consider the pH of the solution to ensure the success of the procedure. If you’re doing DNA purification, keep the pH of the solution between 7.0 and 8.0. However, if you want to obtain purified RNA samples, keep the pH around 4.5.