Most batches of commercial liquified phenol are clear and colorless and can be used in molecular cloning without redistillation. Occasionally, batches of liquified phenol are pink or yellow, and these should be rejected and returned to the manufacturer. Crystalline phenol is not recommended because it must be redistilled at 160°C to remove oxidation products, such as quinones, that cause the breakdown of phosphodiester bonds or cause cross-linking of RNA and DNA.Before use, phenol must be equilibrated to a pH of >7.8 because the DNA partitions into the organic phase at acid pH. Wear gloves, full face protection, and a lab coat when carrying out this procedure.

1. Store liquified phenol at -20°C. As needed, remove the phenol from the freezer, allow it to warm to room temperature, and then melt it at 68°C. Add hydroxyquinoline to a final concentration of 0.1%. This compound is an antioxidant, a partial inhibitor of RNase, and a weak chelator of metal ions. In addition, its yellow color provides a convenient way to identify the organic phase.

2. To the melted phenol, add an equal volume of buffer (usually 0.5 M Tris-Cl [pH 8.0] at room temperature). Stir the mixture on a magnetic stirrer for 15 minutes. Turn off the stirrer, and when the two phases have separated, aspirate as much as possible of the upper (aqueous) phase using a glass pipette attached to a vacuum line equipped with appropriate traps.

3. Add an equal volume of 0. 1 M Tris-Cl (pH 8.0) to the phenol. Stir the mixture on a magnetic stirrer for 15 minutes. Turn off the stirrer and remove the upper aqueous phase as described in Step 2. Repeat the extractions until the pH of the phenolic phase is >7.8 (as measured with pH paper).

4. After the phenol is equilibrated and the final aqueous phase has been removed, add 0.1 volume of 0.1 M Tris-Cl (pH 8.0) containing 0.2% P-mercaptoethanol. The phenol solution may be stored in this form under 100 mM Tris-Cl (pH 8.0) in a light-tight bottle at 4°C for periods of up to 1 month.

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