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Preparation of Equilibrated Phenol The application of molecular biology techniques
to the analysis of complex genomes depends on the ability to prepare pure, high molecular
wt. DNA. For applications requiring a higher pH, such as the isolation of large intact
genomic DNA, addition of the Equilibrated phenol is recommended.
Equilibrated phenol is one such reagent used in nucleic acid purification procedures and
dissolution of protein.
The chemical formula of phenol is C6H5OH and as a solid it is a white crystalline compound.
Its melting point is 41° C and its boiling point is 182° C. Phenol has a sweet odour
that is detectable at 0.06 PPM, which enables it to be used as an air freshener. However,
the chemical is highly corrosive and moderately toxic. Therefore it is also used as a powerful
disinfectant and a germicide.
Procedure: Remove the crystalline phenol from the –20oC
freezer and thaw it at 60-65oC in a water bath.
Add desired volume of phenol to an appropriate sized bottle.
Add an equal volume of 10X TE to the phenol. Shake vigorously using a magnetic stirrer.
Allow the layers to separate. Aspirate off the top aqueous layer. Do this
in the fume hood. Repeat with a second equal volume of 10X TE.
Add an equal volume of 1X TE to the phenol. Repeat with a second equal volume of 1X TE.
Leave a small layer of 1X TE above the phenol after the final aspiration.
Check the pH of the TE by dropping ~10 µl onto pH paper. Its pH should be ~ 8; if it
is still too high, perform additional TE equilibration steps.
Caution:
Phenol is a corrosive. It will cause severe burns if you get it on your skin..Wear gloves,
goggles and a lab coat work in the fume hood when working with large quantities of phenol.
If you spill it on yourself, wash the skin area well with water.