The standard way to remove proteins from nucleic acid solutions is to extract once with phenol and once with chloroform (Sevac). This procedure takes advantage of the fact that deproteinisation is more efficient when two different organic solvents are used instead of one. Furthermore, although phenol denatures proteins efficiently, it does not completely inhibit RNase activity [115]. The final extraction with chloroform also removes any lingering traces of phenol from the nucleic acid preparation.
The sample is diluted to a volume of about 400 to 500 µl. One volume TE-saturated phenol is added, mixed by vortexing4. A centrifugation step is performed to separate layers (room temperature, 2min, 13000 rpm). The upper aqueous layer is removed and transferred to a new tube. The lower organic layer and interface5 is discarded. The aqueous layer is mixed with 0.5 volume Sevac (chloroform:isoamyl alcohol 24:1) and centrifuged for 2 minutes at 13000 rpm. The aqueous layer is removed and collected in a new tube [116, 117].
© 2001 Alexander Binder