Extraction of genomic DNA
Removal of proteins from nucleic acids can be achieved by extraction with phenol:chloroform solutions. For crude mixtures of nucleic acids, digestion with a broad range proteolytic enzyme prior to the phenol:chloroform extraction may be beneficial. Although proteins are efficiently denatured by phenol, RNase activity is not completely inhibited. Therefore, a small amount of isoamyl alcohol is added to further ensure the deactivation of RNase activity. For DNA extraction, the pH of the phenol phase can be adjusted to 8.0 by equilibration with tris buffer.
Procedure
The protocol for removal of proteins form nucleic acids follows:
1. Mix equal volume of phenol: chloroform: isoamyl alcohol solution with the nucleic acid solution in polypropylene tube with a cap. Mix briefly until an emulsion forms.
2. Centrifuge at 12,000 g for 3-5 minutes at room temperature. The aqueous phase (upper) and organic phase (lower) should be well separated. The interface, which typically appears as an opaque disc, contains the denatured proteins.
3. Transfer the aqueous phase to a new tube. Discard the interface and organic phase.
4. Repeat steps 1-3 until no protein is visible at the interface.
5. Mix equal volume of chloroform with the aqueous phase. Centrifuge at 12,000 g for 3-5 minutes. This step will remove any residual phenol. Optional step.
6. Pipette upper phase into a new tube and precipitate the nucleic acid as desired.
Reference
Sambrook, Fritsch, Maniatis, Molecular Cloning: A Laboratory Manual 2nd Edition, Vol. 3, pages E3 – E4; Cold Spring Harbor Laboratory Press 1989