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Preparation of Equilibrated Phenol
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Procedure:

 

  1. Remove the crystalline phenol from the -20°C freezer and thaw it at 60-65°C.
  2.  Add desired volume of phenol to an appropriately sized bottle.
    (If you take 250-500ml of equilibrated phenol, use a 1000ml bottle.)
  3. Add equal volume of 10X TE to the phenol.
  4. Shake vigorously and allow the layers to separate.
  5. Remove the aqueous (top) layer. Do this using a fume hood.
  6. Repeat the process by adding equal volume of 10X TE again.
  7. Add equal volume of 1X TE buffer  to the phenol.
  8. Repeat the process by adding equal volume of 1X TE buffer again.
  9. After the final aspiration, leave a small layer of 1X TE buffer above the phenol .
  10. Evaluate  the pH of the TE buffer by dropping ~10 μl onto pH paper. It should be ~pH 8; when it is still.

 

Recipes:

 

TE buffer:

 

  • 10 mM Tris-Cl, pH 7.5.
  • 1 mM EDTA.


        To make TE Buffer from 1M stock of Tris-Cl (pH 7.5) and 500 mM stock of EDTA (pH 8.0),  Take 10ml 1M Tris-Cl pH 7.5 and 2ml 500mM EDTA.

 

1M Tris (crystallized free base):

 

  • Tris(hydroxymethyl) aminomethane.
  • FW 121.4 g/mol.
  • 60.57 g in 0.5L  Distilled water.
  • Adjust pH to 7.5 using HCl.

 

0.5M EDTA:

 

  • Diaminoethane tetraacetic acid.
  • FW 372.2 g/mol.
  • 18.6 g in 100ml Distilled  water.

          Adjust pH to 8.0 using NaOH otherwise EDTA will not soluble .Use vigorous stirring, moderate heat (if desired) and time.

 

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