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Aqueous samples, lysed cells, or homogenised tissue are mixed with equal volumes of a phenol:chloroform mixture. This mixture is then centrifuged. Because the phenol:chloroform mixture is immiscible with water, the centrifuge will cause two distinct phases to form: an upper aqueous phase, and a lower organic phase.
Chloroform: Chloroform is stabilized with small quantities of amylene or ethanol, because exposure of pure chloroform to oxygen and ultraviolet light produces phosgene gas. Some chloroform solutions come as pre-made a 96% chloroform, 4% isoamyl alcohol mixture that can be mixed with an equal volume of phenol to obtain the 25:24:1 solution.
In the author's own words, "The first phase in the reaction is probably the formation of acetonechloroform--(which may, indeed, be used in place of the chloroform), this being then acted on by sodium hydroxide in presence of acetone, yielding α-hydroxyisobutyric acid, which, with the phenol, gives α-phenoxyisobutyric acid. The chloroform may ...
A commonly used method is guanidinium thiocyanate-phenol-chloroform extraction. It is not strictly necessary to use phenol or chloroform if extracting RNA for Northern blotting or DNA for Southern blot analysis because the gel electrophoresis followed by transfer to a membrane will separate the RNA/DNA from the proteins. Additionally, since ...
The correct name of the method is guanidinium thiocyanate-phenol-chloroform extraction. The use of TRIzol can result in DNA yields comparable to other extraction methods, and it leads to >50% bigger RNA yield. [5] [6] An alternative method for RNA extraction is phenol extraction and TCA/acetone precipitation. Chloroform should be exchanged with ...
Chloroform: 1.48 61.2 3.88 –63.5 –4.90 ... Phenol: 181.75 3.60 43.0 –7.27 K f [2] K b [1] Water: ... Trifluoroacetic Acid: 71.8 [30] Dioxane: 1.03 101.0 11.8 ...