Buffer-saturated Phenol - (Apr/03/2012 )
How can I prepare a 38% bufer-saturated phenol (v/v) solution? I need it for an Extraction buffer to isolate RNA.
You can buy it quite easily - many companies sell it, but the most reliable and quickest is Sigma-Aldrich. I would strongly recommend buying it!!!!
Making it is time consuming and relatively dangerous (phenol is toxic, hard to remove, and causes burns). However if you want to proceed with making it, equilibration of phenol is performed by:
1: Remove liquid phenol from freezer and melt at 68 deg C, add hydroxyquinolone to 0.1% (yellow colour gives convenient way to identify organic phase, + is an antioxidant)
2: add equal volume of 0.5 M tris-Cl (ph 4.5) and stir for 15 min. Allow phases to separate and remove upper aqueous layer into appropriate discard (NOT DOWN THE SINK!!!!!)
3: Add equal volume 0.1 M tris-cl, pH 4.5, and stir for 15 min, remove aqueous phase. repeat until pH is 4.5, by measuring the pH of th buffer after each equilibration.
4: when finally equilibrated, add 0.1 volumes of tris-cl, pH 4.5, with 0.2% 2-mercaptoethanol and store in a light proof bottle at 4 deg C for 1 month.
After this time, equilibration will need to be repeated to remove oxidation products.