Neutralization and Equilibration of Phenol
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Lab Protocol |
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Malaysia University |
Neutralization and Equilibration of Phenol
Contributor:
The Laboratory of Donald Rio at the University of California, Berkeley
Procedure
1. Remove a 1-liter bottle of Phenol from the freezer and thaw overnight at room temperature.
2. OPTIONAL: Add 8-Hydroxyquinoline to the Phenol to a final concentration of 0.1% (w/v) and mix well (see Hint #1).
3. Aspirate off any of the top phase (aqueous phase) that may be present until approximately 1 cm is left.
4. Add 100 ml of 0.3 M Tris Base, shake extensively, and let the phases separate (approximately 30 min) at room temperature.
5. Aspirate off the top aqueous phase.
6. Add 100 ml of TE Buffer, shake extensively, and let the phases separate (approximately 30 min) at room temperature.
7. Check the pH of the aqueous phase; it should be between pH 7 and 8.
8. Aspirate off the top aqueous phase.
6. Repeat Step #6 to #8 (TE saturation) two more times.
7. Store only the Phenol with approximately 1 cm of TE Buffer layer on top at 4°C in a brown glass bottle.
Solutions
TE Buffer
10 mM Tris
pH 8.0
1 mM EDTA 
Tris Base
0.3 M Tris base
BioReagents and Chemicals
8-Hydroxyquinoline
Phenol
EDTA
Tris Base
Protocol Hints
1. 8-Hydroxyquinoline is an anti-oxidant as well as a colorizing agent. Coloring the Phenol with 8-Hydroxyquinoline will make extraction procedures easier since the Phenol will be readily identifiable.
