Protocol for Ultracentrifugal RNA Virus Purification
The following protocol has been established for rotavirus purification, but may also be applicable for other RNA viruses . A floor-type ultracentrifuge such as Beckman Coulter's Optima ultracentrifuge is recommended for use with this protocol.
Divide 1 L of a virus-containing culture into 6 bottles.
Centrifuge using a JA-14 fixed-angle rotor in a high performance centrifuge at 10,000 rpm (15,000 x g) for 10 minutes to pellet cell fragments/debris.
Transfer 150 mL of the supernatant to each of 6 tubes.
Centrifuge using a JA-14 fixed-angle rotor in a high performance centrifuge at 14,000 rpm (30,000 x g) for 14-16 hours to pellet virus. Alternatively, process 300 mL at a time with a Type 45 Ti fixed-angle rotor in an ultracentrifuge by centrifuging at either 30,000 rpm for 2 hours or 40,000 rpm for 1.5 hours.
Remove the supernatant and suspend the pellets with 26 mL of PBS per tube.
Place 4.5 mL of 70% sucrose solution at the base of a 38.5 mL ultracentrifuge tube and overlay it with 6.5 mL of 30% sucrose solution. Then layer 26 mL of the virus suspension on top.
Centrifuge using a SW 32 Ti swinging bucket rotor in an ultracentrifuge at 32,000 rpm (175,000 x g) for 2 hours.
Extract the white band (virus crude fraction) above the 70% sucrose solution.
Place 55% (w/v) cesium chloride solution at the base of a 5 mL ultracentrifuge tube and overlay it with an equal quantity of 40% (w/v) cesium chloride solution. Then layer 0.5-0.8 mL of virus crude fraction on top.
Centrifuge using a SW 55 Ti swinging bucket rotor in an ultracentrifuge at 46,000 rpm (257,000 x g) for 16-20 hours.
Extract the blue bands (purified virus fragments).
While the above suggested protocol has been tested to be effective, if you are instead looking for a convenient solution for RNA isolation, complete with all buffers and reagents required, we recommend looking at the variety of viral & total RNA extraction kits that we have to offer:
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