hotphenolextraktion

Hot phenol extraction is a laboratory technique used to separate nucleic acids from proteins and other cellular components by using phenol heated to near boiling under acidic conditions. In this approach, a cell lysate is mixed with hot phenol and then partitioned by centrifugation. Under elevated temperature and acidic pH, proteins denature and preferentially migrate into the organic phase, while nucleic acids largely remain in the aqueous phase. The aqueous phase can then be further purified by a phenol–chloroform extraction to remove residual contaminants, followed by ethanol or isopropanol precipitation to recover RNA (or DNA, depending on the specific protocol).

Procedure overview: cells or tissues are lysed in an appropriate buffer and mixed with hot phenol (about 65–70°C, pH around 4.3). The mixture is centrifuged to separate phases, and the upper aqueous phase containing the nucleic acids is recovered. A subsequent organic extraction with phenol–chloroform–isoamyl alcohol can increase purity, after which nucleic acids are precipitated, washed, and resuspended for downstream analysis. The method is often used to enrich RNA and to minimize RNase activity during extraction, with additional DNase steps applied if DNA-free RNA is required.

Applications and context: hot phenol extraction has historically played a central role in RNA purification, especially for difficult or RNase-rich samples. It remains less common in routine workflows, having been largely supplanted by silica-column and magnetic bead-based kits, but it is still used in certain plant, bacterial, or recalcitrant samples where alternative methods are less effective. Safety and handling considerations are important due to the corrosive and toxic nature of phenol; procedures are conducted in a fume hood with appropriate PPE and waste disposal.

See also: phenol–chloroform extraction, RNA purification.