WinterVomitingLab:Protocols/Organic solvent extraction
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Purification of non-enveloped viruses from a dirty sample.
Choose one of the following:
- Chloroform (CHCl3)
- Vertrel XF (DuPont)
- 1,1,1,2,3,4,4,5,5,5-Decafluoropentane (C5H2F10)
- Freon: trichlorofluoromethane (CCl3)
- Freon is no longer available commercially due to its high potential for ozone depletion. Vertrel® XF is now recommended as an “environmentally friendly” alternative solvent (Mendez et al, 2000).
- Add an equal volume of organic solvent (chloroform, Freon, Vertrel®XF) to sample (v:v).
- Vortex until thoroughly mixed.
- Centrifuge for 15 min at 2,500 x g (for large tubes) or 10 min at 13,000 rpm in a microcentrifuge.
- Carefully remove from centrifuge without disturbing the separation of the organic and aqueous phases.
- Collect the aqueous phase (top) containing viruses taking care not to disturb the interphase. Note: the interphase may contain a portion of the viruses, particularly if the sample was very turbid.
- Consider performing a second chloroform extraction on the interphase, adding the derived supernatant to the aqueous portion previously collected.
- Dispose organic solvent phases in hazardous materials collection container.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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