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