Preparation of Tris-saturated Phenol

Overview:

• Phenol is a colorless crystalline solid. Phenol gradually turns pink to brownish color due to oxidation upon exposure to air and light. 
• Oxidation products (e.g., quinones) of phenol can cause DNA damage (the breakdown of phosphodiester bonds, cross-linking of nucleic acids), therefore, they should be removed from the phenol by redistillation.
• Redistillation of phenol at 182°C under nitrogen removes oxidized products from the phenol. Redistilled phenol should be frozen and kept in a dark bottle.
• In order to utilize phenol for extraction of the nucleic acids (e.g., DNA and RNA), phenol is first equilibrated with buffer (Tris. Cl, pH 8.0) or water.
• Tris-saturated phenol which has pH ≈8.0 is utilized for the isolation and purification of DNA.
• To prepare Tris-saturated phenol, phenol is equilibrated with Tris.Cl (pH 8.0) solution until the pH of phenol reaches ≈8.0.
• Often a small amount of 8-hydroxyquinoline (0.1%) is added to the phenol. 8-hydroxyquinoline is an antioxidant, which prevents oxidation of phenol. In addition, its yellowish color also helps to identify the phenolic phase from the aqueous phase during the extraction process.
• When Tris-saturated phenol or Phenol:chloroform (1:1) solution is used to extract biological samples or DNA solution, both DNA and RNA partitions into the aqueous phase, leaving most impurities like protein and lipids in the phenolic (organic) phase or at the interface.
• If required, RNA contamination from the extracted DNA can be removed by RNase A digestion.

Requirements

Reagents and solutions
♦ Redistilled Phenol (molecular biology grade: Stored in aliquots at -20℃)
♦ 8-hydroxyquinoline
♦ 0.5 M Tris.Cl buffer (pH 8.0)
♦ 0.1 M Tris.Cl buffer (pH 8.0)

Equipment and disposables
♦ Fume Hood
♦ Magnetic stirrer and Magnetic stir bar
♦ Glass Bottle/Beaker
♦ Measuring cylinder

Objective
Preparation of Tris.Cl (pH 8.0) saturated Phenol

Precautions:
1. Phenol is volatile and caustic. Care must always be taken when handling phenol (wear lab coat, gloves, and eye protection). Do all operations in a fume hood.
2. Discard the waste according to your institution’s waste-disposal guidelines.
3. Avoid exposure of phenol to light. Cover the bottle/beaker containing phenol with aluminum foil.

Prior to start:
Set the water bath at 50°C in a fume hood.

Procedure:

Step 1: Thaw the frozen phenol by placing the bottle in a 50°C water bath.

Step 2: Transfer 100 ml phenol to a beaker / bottle.

Step 3: Add ≈0.1 gram 8-hydroxyquinoline [final conc ≈0.1% (w/v)]. Mix to dissolve it.

Step 4. Add 100 ml of 0.5 M Tris.Cl (pH 8.0) to the phenol. Stir the mixture for 15 – 30 min on a magnetic stirrer at room temperature. Place the bottle/beaker in a 50°C water bath and allow phase separation. Discard the aqueous phase. Repeat this step (3 – 4 times) until the pH of the aqueous phase is >7.8.

Step 5: Now transfer the tris-saturated phenol to a glass bottle (100 ml) and add ≈20 ml 0.1M Tris.Cl over the phenol. Tris.Cl will form a thin upper layer (0.5 -1 cm).

Storage:
The Tris.Cl saturated phenol can be stored in the dark at 4°C for 3 – 6 months. Periodically check the pH of phenol during storage. Discard it if the pH of the phenol is <7.5.