Application of Prepared Liquid Phenols

Due to the corrosive nature of phenol, preparation of phenol solutions for use in molecular biology is a time-consuming and often hazardous procedure. Frequently it is necessary to redistill the phenol prior to use to remove contaminants and oxidation products that can damage nucleic acids. In some cases, the pH of phenol solutions needs to be adjusted before use. Safety precautions such as protective eyewear, gloves, and a fume hood are necessary. Ambion's range of pre-mixed, quality tested, saturated phenols eliminates these handling problems, and offers a more convenient, safer, and easy alternative to preparing solutions from crystalline phenol.

Preparation of Nucleic Acids

Efficient extraction of cell extracts or solutions containing nucleic acid are most often performed with a series of phenol and phenol:chloroform extractions at a specific pH. Both phenol and chloroform cause proteins to become denatured and become soluble in the organic phase or interphase, while nucleic acids remain in the aqueous phase. After centrifugation, the aqueous phase containing nucleic acid is re-extracted with an equal volume of chloroform:isoamyl alcohol (1,3,4). This combination of extractions is thought to reduce the loss of RNA due to the formation of insoluble protein:RNA complexes at the interphase.

Chloroform is mixed with phenol to increase the efficiency of nucleic acid extractions by reducing losses of RNA at the interphase. The increased efficiency is due to chloroform's ability to denature proteins and aid in the removal of lipids, thus improving separation of nucleic acid into the aqueous phase. Phase separation is also enhanced, which assists in the removal of the aqueous phase with minimal cross contamination from the organic phase. Often isoamyl alcohol is added to phenol:chloroform to reduce foaming.

Determining the pH of Phenol

Depending on the pH of the phenol, DNA will partition into either the organic phase or the aqueous phase (5,6). Thus, it is necessary to accurately determine the pH of phenol solutions. However, accurate pH measurements of organic phenol and phenol:chloroform can difficult to achieve (2). Standard reference electrodes measure the liquid junction potential between the electrode's potassium chloride filling solution and the sample. Organics such as phenol and chloroform have very low dielectric constants compared to water. A very large liquid junction potential can cause problems such as pH drift, long stabilization times and damage to the pH electrode. Because of this, pH paper has often been used to measure the pH of phenol solutions, however, phenol destroys the indicator chemical of the pH paper, resulting in inaccurate pH measurement.

To accurately measure the pH of saturated phenol, it is necessary to solubilize the phenol in an aqueous medium. The following method is used at Ambion to determine pH of phenol solutions:

For phenol:chloroform:IAA or acid phenol:chloroform solutions - mix 2 ml of the organic phase with 8 ml of methanol and 10 ml of water. Measure the pH of the entire sample.
For saturated phenols - mix 2 ml of the organic phase with 5 ml of methanol and 13 ml of water. Measure the pH of the entire sample.

Which Ambion Phenol to Choose?

In order to choose the phenol solution best suited for your application, it is necessary to have an understanding of the effect of pH on the function of phenol during nucleic acid isolation. DNA partitioning is pH dependent; at pH 7.0 or higher, both DNA and RNA partition into the aqueous phase. At an acidic pH, below pH 7.0, DNA will be denatured and partition into the organic phase and interphase, leaving the RNA alone in the aqueous phase. For most DNA isolation procedures, a solution of phenol:chloroform:isoamyl alcohol (25:24:1) at pH 8.0 is the phenol solution of choice.

For RNA isolation free of most contaminating DNA, an acidic phenol or phenol:chloroform solution is required. The chloroform will help reduce the loss of messenger RNA at the interphase due to insoluble protein:RNA complexes. Ambion's Acid Phenol:Chloroform at pH 4.7 (5:1 ratio of phenol:chloroform) is recommended. (Note that while small amounts of contaminating genomic DNA in total RNA will not compromise the validity of the results obtained from Northern blotting and nuclease protection assays, it is critical that such contamination be removed for RT-PCR-based assays. Ambion offers both kits and reagents to assist in DNA removal from RNA samples.

Quality Control

Ambion does extensive quality control testing of all reagents. All of our phenol products are assayed for the presence of ribonucleases and endonucleases in a two-hour long continuous shaking incubation. All reagents are subject to an additional level of quality control in that they are used routinely in Ambion's research labs.

Ambion's prepared liquid phenols offer a convenient, safe and easy alternative to preparing solutions from crystalline phenol. All phenols are packaged under inert gas and are stable for > 6 months when stored unopened at -20° C. If the product will be frequently opened, aliquotting into 50 ml tubes and storing all aliquots (except the one in immediate use) at -20° C will prolong the storage life for up to one year.

References

1. Ausubel, F.A., Brent,R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., (1987) Current Protocols in Molecular Biology.
2. Kleinhenz, E.A., and Cohen, S.B. (1991) Biotechniques. 10: 740.
3. Wallace, D.M. (1987) Methods in Enzymology 152: 33.
4. Sambrook, J., Fritsch, E. F., Maniatis, T., (1989) Molecular Cloning: A Laboratory Manual 2nd Edition.
5. Perry, R. P., Torre, J. La, Kelly, D. E., and Greenberg, J. R. (1972) Biochem. Biophys. Acta 262, 220.
6. Brawerman, G., Mendecki, J., and Lee, S. Y. (1972) Biochemistry 11, 637.
7. Chirgwin, John M. Przybyla, Alan E., MacDonald, Raymond J., and Rutter, William, J. (1979) Biochemistry 18:5294.
8. Chomczynski P., Sacchi, N.: (1987) Anal Biochem 162:156.

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