Phenol-chloroform extraction with ethanol precipitation
To the viral suspension (≤0.6 mL per 2-mL microcentrifuge tube, scale up for larger volumes) add an equal volume of PCI and shake to emulsify.
Centrifuge at 10,000g for 5 min to facilitate separation of the organic and aqueous phases.
Transfer the DNA-containing aqueous phase (upper) to a new tube by aspiration with a pipette, being careful to avoid material at the interface.
Repeat steps 1–3 as needed until the interface appears to be free of extracted material (one extraction may suffice for relatively pure viral preparations).
Add an equal volume of CI to the aqueous phase and shake to emulsify.
Centrifuge as in step 2 to separate phases.
Transfer the aqueous phase (upper) to a new tube.
Add 1 µL polyacryl carrier.
Add 1/10 of a volume of sodium acetate and invert tube or vortex to mix.
Add 2 volumes of ethanol and invert tube to mix.
Incubate sample on ice for 10 min.
Centrifuge for 10 to 30 min, at 0–4°C if possible.
Aspirate or decant the supernatant, being careful not to disturb the pellet.
Add 500 µL ice-cold 70% ethanol.
Centrifuge at 10,000g for 10 min.
Decant or aspirate supernatant as completely as possible, being careful not to disturb the pellet.
Allow residual liquid in the tube to evaporate by air-drying with the cap open and the tube upside down or by placing briefly in a centrifugal vacuum concentrator (e.g., SpeedVac concentrator, Thermo Scientific; Concentrator plus, Eppendorf).
Resuspend the dried pellet in a small volume of Tris (10 mM, pH 8) or TE buffer.
The purified, solubilized DNA may be stored at 4°C for short periods of time, at –20°C for long periods of time, and at –80°C indefinitely.
