Summary: Buffered Phenol
Why buffer phenol?
Buffered phenol more than 2 months old can damage DNA and interfere with cloning!
1. Start with a 500 g bottle of ultrapure phenol. Cover phenol with ~200 ml dH2O. Place in 37°C
water bath. Swirl intermittantly. Water will mix with, and liquify, the phenol.
2. Add a large stir bar. Add 0.5 g 8-hydroxyquinoline to 0.1%. Mix to obtain a fine emulsion.
3. Close cap tightly. Seal with parafilm. Place at 4°C. Let phases separate completely (overnight).
4. Aliquot liquefied phenol into 50 ml red-cap tubes. Add ~35 ml of phenol to each tube. Cover
phenol with ~3-5 ml dH2O. Wrap caps with Parafilm. Store at -20°C.
1. Thaw liquefied phenol at 25-37°C. Process 2-4 tubes of phenol at a time.
2. Prepare 50 ml of 500 mM Tris, pH 8: 2.1 g Tris-HCl + 1.4 g Tris Base in 50 ml dH2O.
3. Add 10 ml 500 mM Tris, pH 8 to each tube. Shake thoroughly. Spin to separate phases. Carefully
remove upper aqueous layer with aspirator.
4. Prepare 50 mM Tris, pH 8 by diluting 500 mM Tris, pH 8 solution.
5. Add 10 ml 50 mM Tris, pH 8 to each tube. Shake thoroughly. Spin. Remove upper layer.
6. Add 10 ml TE (pH 8) to each tube. Shake thoroughly. Spin. Remove upper layer.
7. Pour phenol into Pyrex glass bottle with orange cap. Top off with 10 ml TE (pH 8) plus 0.1%
(vol/vol) ß-mercaptoethanol. Store equilibrated phenol in dark at 4°C.