Chloroform phenol is a standard laboratory reagent used to extract DNA and RNA from biological samples. Making it involves equilibrating crystalline phenol to the correct pH, then combining it with chloroform in the proper ratio. The process takes a few hours, mostly waiting for phases to separate, and requires careful safety precautions since both chemicals are hazardous.
How the Reagent Works
When you mix a cell sample with phenol:chloroform and spin it in a centrifuge, the solution separates into layers. Proteins and cellular debris migrate into the lower organic phase, while nucleic acids (DNA or RNA) stay dissolved in the upper water-based phase. You then pipette off that top layer to collect your purified nucleic acids. The chloroform in the mixture sharpens the boundary between the two phases, making it easier to collect the aqueous layer without dragging along contaminants.
The pH of the phenol determines what you extract. For DNA, you equilibrate phenol to a neutral or slightly basic pH of 7 to 8. At this pH, DNA stays in the aqueous phase where you can recover it. For RNA extraction, acidic conditions (around pH 4 to 4.5) keep RNA in the aqueous phase while pushing DNA into the organic layer. Getting the pH right is the single most important step in preparing the reagent.
Equilibrating the Phenol
Laboratory-grade phenol typically comes as a 100 g bottle of crystalline solid. The first step is melting and buffering it, a process called equilibration. Work in a fume hood for all of these steps.
- Melt and buffer. Open the phenol bottle in the fume hood and add roughly 100 ml of 50 mM Tris-HCl at pH 8. Close the lid tightly and shake gently. Let it stand for one to two hours until the phenol liquefies and the two phases separate clearly.
- Remove the supernatant. Pipette off the upper aqueous layer and discard it into a chlorinated solvent waste container.
- Wash repeatedly. Add another 100 ml of 50 mM Tris-HCl at pH 8, shake gently, and let the phases separate for a few minutes. Repeat this wash four or five times until the pH of the upper layer reads between 7 and 8 on pH strips.
- Aliquot and store. Divide the equilibrated phenol into 10 to 20 ml portions in Falcon tubes. Add 10 ml of 50 mM Tris-HCl pH 8 on top of each aliquot. Store under this Tris buffer layer at negative 20°C.
The Tris buffer overlay prevents the phenol from oxidizing during storage. Oxidized phenol turns pink or brown and produces quinones that damage nucleic acids, so never use discolored phenol.
Mixing the Final Reagent
The standard formulation is phenol:chloroform:isoamyl alcohol at a 25:24:1 ratio by volume. To prepare it, combine 25 volumes of your equilibrated phenol with 24 volumes of chloroform and 1 volume of isoamyl alcohol. The isoamyl alcohol reduces foaming during mixing and helps stabilize the interface between phases during extraction.
For a typical working batch, you might mix 25 ml equilibrated phenol, 24 ml chloroform, and 1 ml isoamyl alcohol. Mix gently by inversion rather than vortexing. Store the finished reagent at 4°C under a layer of Tris buffer in a light-protected container, since both phenol and chloroform degrade with light exposure.
Adding 8-Hydroxyquinoline
Many protocols call for adding 8-hydroxyquinoline to the phenol at a final concentration of 0.1% by weight. This additive serves several purposes at once. It acts as an antioxidant, slowing the formation of quinones that would otherwise accumulate as the phenol oxidizes. It partially inhibits RNase enzymes, which is useful when extracting RNA. It also chelates heavy metals that can degrade nucleic acids over extended incubations.
Perhaps its most practical benefit: 8-hydroxyquinoline turns the organic phase bright yellow, making it much easier to visually distinguish the organic layer from the aqueous layer when pipetting. Without it, the two phases can look similar, and accidentally pulling from the wrong layer is one of the most common extraction mistakes.
Safety Requirements
Phenol causes severe chemical burns on skin contact and is readily absorbed through the skin. Chloroform is toxic by inhalation and a suspected carcinogen. Both chemicals demand serious precautions.
All work with stock phenol solutions and with the mixed reagent should be done inside a fume hood. Even dilute phenol warrants a fume hood because the sash provides splash protection and the ventilation contains vapors, especially if a spill occurs.
Glove selection matters more here than with most lab chemicals. Standard nitrile gloves are not recommended for phenol work. For concentrations above 70%, use butyl rubber, Viton, or Silver Shield gloves. Neoprene and polyvinyl alcohol gloves work for shorter tasks (one to four hours of resistance) but should be thicker than 0.3 mm. If you use thin disposable gloves, treat them as splash protection only and remove them immediately if phenol contacts the surface. Double gloving with heavy-weight disposables (0.2 mm or thicker) is good practice. For chloroform-phenol mixtures specifically, butyl rubber provides over 90 minutes of breakthrough resistance.
If phenol contacts your skin, the recommended first aid is to apply polyethylene glycol 300 or 400 (PEG-300 or PEG-400) rather than water. Many labs keep a bottle of PEG near the fume hood for this purpose. Water alone does not effectively remove phenol from skin.
Checking Extraction Quality
After using your reagent to extract nucleic acids, you can verify purity with a spectrophotometer. The ratio of absorbance at 260 nm to 280 nm indicates whether protein or phenol contamination carried over. For pure DNA, this ratio should be around 1.8. Values at or below 1.6 suggest protein or residual phenol in the sample.
A secondary check is the 260/230 ratio, which flags contaminants like salts, carbohydrates, or leftover phenol. Pure DNA typically falls between 2.0 and 2.2. Low values here often point to phenol carryover, which means the aqueous phase was not cleanly separated during extraction.
Common Problems and Fixes
Poor phase separation is the most frequent issue. If the boundary between layers is indistinct or the phases do not fully separate, the sample may have too much detergent or the volumes may be unbalanced. Ensuring the correct 25:24:1 ratio and centrifuging at adequate speed typically resolves this.
Contamination from the interphase is another common problem. The interphase sits between the aqueous and organic layers and contains denatured proteins and cellular debris. When pipetting the aqueous layer, avoid dipping into or disturbing this boundary. Any material pulled from the interphase will carry protein and phenol into your purified sample, lowering your 260/280 ratio and potentially interfering with downstream applications. Taking slightly less of the aqueous phase is always better than risking interphase contamination.
Phenol that has turned pink, brown, or dark should be discarded. Oxidized phenol contains quinones that nick and degrade nucleic acids. Proper storage under Tris buffer at negative 20°C, with 8-hydroxyquinoline added, prevents this for several months.