WinterVomitingLab:Protocols/Ultracentrifugation: Difference between revisions
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(New page: Return to WinterVomitingLab ==Overview== Virus concentration by ultracentrifugation (pelleting). ==Materials== * Virus-containing sample * Ultracentrifuge * Ultracentrifuge tubes * ...) |
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## syringe filtration through 0.45 and/or 0.22 uM filters | ## syringe filtration through 0.45 and/or 0.22 uM filters | ||
## [[WinterVomitingLab:Protocols/Organic solvent extraction|organic solvent extraction]] | ## [[WinterVomitingLab:Protocols/Organic solvent extraction|organic solvent extraction]] | ||
# 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. | |||
# Load samples into ultracentrifuge rotor, ensuring proper balancing. | |||
# Place rotor in the ultracentrifuge, close the lid, and turn on the vacuum. | |||
# Centrifuge at 100K x g (35,500 rpm using the NVT-90 rotor) for 1 hr at 4°C. | |||
# At the end of the run, release the vacuum, remove rotor and carefully remove samples. | |||
# Remove and discard the supernatant. For Quick Seal tubes, this should be done using a syringe and needle. | |||
# Add buffer of choice (such as PBS) at the desired volume to the tube over the pellet. | |||
## 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). | |||
## Recover the concentrated virus and store at -70°C. | |||
==Notes== | ==Notes== | ||
Latest revision as of 20:25, 18 September 2013
Return to WinterVomitingLab
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
- At least 1-2 hours before beginning, cool the centrifuge and the rotor to 4°C.
- If there are a lot of particulates in the virus-containing sample, consider at least one of the following:
- centrifugation at 9,000 x g for 15 min, retaining the supernatant
- syringe filtration through 0.45 and/or 0.22 uM filters
- organic solvent extraction
- 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.
- Load samples into ultracentrifuge rotor, ensuring proper balancing.
- Place rotor in the ultracentrifuge, close the lid, and turn on the vacuum.
- Centrifuge at 100K x g (35,500 rpm using the NVT-90 rotor) for 1 hr at 4°C.
- At the end of the run, release the vacuum, remove rotor and carefully remove samples.
- Remove and discard the supernatant. For Quick Seal tubes, this should be done using a syringe and needle.
- Add buffer of choice (such as PBS) at the desired volume to the tube over the pellet.
- 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).
- 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!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- 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
