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 .
