Deproteination using phenol/chloroform (Maniatis et al., 1982)'Phenol' as used is Analar grade. Phenol should be melted at 65°C,
8-hydroxyquinoline added to a final concentration of 0.1%, and equilibrated three times with an equal volume of 1M Tris.HCl, pH 7.0.
The final Tris wash is replaced with TE (10mM Tris, 1mM EDTA, pH8.0) and the phenol stored in the dark at 4°C. 8-hydroxyquinoline is added to prevent oxidation of the phenol and to act as an inhibitor of RNases.
'Chloroform' as used is a water saturated 24:1(v/v) mixture of chloroform and isoamyl alcohol. The presence of isoamyl alcohol is required to aid separation of the two phases during extraction of aqueous solutions with chloroform. Phenol:chloroform is a 1:1(v/v) mixture of the above two reagents.
N.B.: Phenol is highly toxic. Gloves should be worn at all times when handling solutions/mixtures containing phenol. Chloroform is a hepatic (liver) toxin and alcohol is flammable.
Nucleic acids are purified as follows:-
1) Add an equal volume of phenol:chloroform to the mixture to be cleaned and vortexed to emulsify the 2 phases.
2) The phases are separated by centrifugation at 13,000 rpm for 2 minutes in a microfuge or 5 minutes at 3,000g in a MSE Centaur for larger volumes.
3) The aqueous upper phase is removed to a clean tube, taking care to avoid any interface material, and the extraction repeated until no interface material is visible.
4) The aqueous phase is then extracted once with an equal volume of chloroform and the nucleic acids alcohol precipitated.
If precipitation is unnecessary the aqueous phase is extracted once with an equal volume of water saturated diethyl ether. The upper ether layer is discarded and any residual ether is removed by heating the sample to 65°C for 5 minutes.
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