WinterVomitingLab:Protocols/Ultracentrifugation

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Contents

Overview

Virus concentration by ultracentrifugation (pelleting).

Materials

  • Virus-containing sample
  • Ultracentrifuge
  • Ultracentrifuge tubes
  • suspension buffer (PBS)

The following may also be needed

  • syringes
  • syringe needle
  • 0.45 and/or 0.22 uM syringe filters


Procedure

  1. At least 1-2 hours before beginning, cool the centrifuge and the rotor to 4°C.
  2. If there are a lot of particulates in the virus-containing sample, consider at least one of the following:
    1. centrifugation at 9,000 x g for 15 min, retaining the supernatant
    2. syringe filtration through 0.45 and/or 0.22 uM filters
    3. organic solvent extraction
  3. Load sample into ultracentrifugation tube. For Quick Seal tubes, use a syringe and needle to top-load the sample. Top off with PBS if needed, so that the tube is completely filled with no air bubbles. For Quick Seal tubes, heat-seal the tubes.
  4. Load samples into ultracentrifuge rotor, ensuring proper balancing.
  5. Place rotor in the ultracentrifuge, close the lid, and turn on the vacuum.
  6. Centrifuge at 100K x g (35,500 rpm using the NVT-90 rotor) for 1 hr at 4°C.
  7. At the end of the run, release the vacuum, remove rotor and carefully remove samples.
  8. Remove and discard the supernatant. For Quick Seal tubes, this should be done using a syringe and needle.
  9. Add buffer of choice (such as PBS) at the desired volume to the tube over the pellet.
    1. Careful that the buffer covers the area of the pellet, place the tube at 4°C for 2 or more hours to allow the pellet to dissolve. (We often leave it over night to dissolve).
    2. Recover the concentrated virus and store at -70°C.

Notes

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

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books


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