Phenol extraction of proteins:
Phenol extraction is frequently used to remove proteins from nucleic acid solutions. A common protocol is to add an equal volume of buffer-saturated phenol or phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v) to an aqueous nucleic acid solution, vortex, and centrifuge at 14,000 x g for 1 min to separate the phases.
Studies at Thermo Fisher Scientific have shown that the concentration of NaCl in the aqueous solution should not exceed 0.5 M for good recovery of DNA. Residual phenol can be removed from the aqueous phase by extraction with an equal volume of chloroform or ether. After extraction, DNA is usually precipitated with ammonium acetate and ethanol as described in another protocol on this server. Ref. Karger, B. D. (1989) FOCUS 11, 14.
A good source of general information on the properties of phenol can be found in Wallace, Donald M. “Large and Small-Scale Phenol Extractions”. Methods in Enz. Volume 152 guide to Molecular Cloning Techniques. 1987. Academic Press, Inc. Berger and Kimmel, eds. Chap.4, pg 33-41.
(a) At pH 5 to 6 DNA is selectively retained in the organic phase and interphase, leaving RNA in the aqueous phase. Therefore a pH greater than 7 is needed if DNA is to be extracted.
(b) At pH values below 7.6, poly A+ RNA is lost to the organic phase if chloroform is not present.
(c) Optimal RNA yields in phenol extraction are obtained if the salt concentration is less than 0.15 M NaCl. Salt concentration in the sample is not a factor for larger DNA molecules.
To store RNA after extraction use DEPC-treated water.
