WinterVomitingLab:Protocols/Organic solvent extraction

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Overview

Purification of non-enveloped viruses from a dirty sample.

Materials

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).

Procedure

  1. Add an equal volume of organic solvent (chloroform, Freon, Vertrel®XF) to sample (v:v).
  2. Vortex until thoroughly mixed.
  3. Centrifuge for 15 min at 2,500 x g (for large tubes) or 10 min at 13,000 rpm in a microcentrifuge.
  4. Carefully remove from centrifuge without disturbing the separation of the organic and aqueous phases.
  5. 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.
  6. Consider performing a second chloroform extraction on the interphase, adding the derived supernatant to the aqueous portion previously collected.
  7. Dispose organic solvent phases in hazardous materials collection container.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
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References

Relevant papers and books

  1. Mendez II, Hermann LL, Hazelton PR, and Coombs KM. A comparative analysis of freon substitutes in the purification of reovirus and calicivirus. J Virol Methods. 2000 Oct;90(1):59-67. DOI:10.1016/s0166-0934(00)00217-2 | PubMed ID:11011081 | HubMed [Paper1]

Contact

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