Brennan:Partial purification of vaccinia virus
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Overview
This protocol is designed for partial purification of vaccinia virus, based on an initial infection of ten 150 mm plates of BSC40 cells.
Materials
Solutions
- Ten 150 mm plates of BSC40 cells, infected with the virus of interest and at full CPE (MOI = 0.01, ~3 days postinfection)
- 1X PBS, 4°C
- 10 mM TRIS.CL, pH 9.0
- 36% sucrose in 10 mM TRIS.CL, pH 9.0
- 1 mM TRIS.CL, pH 9.0
Equipment
- Sterile rubber policeman
- Sterile Dounce homogenizer with tight pestle (A)
- Prechilled Ultra centrifuge SW41 rotor and buckets
Procedure
- Scrape infected cells from dish, using a rubber policeman if necessary, and combine the cells in 50 mL tubes.
- Pellet cells at 900g x 10 minutes.
- Aspirate supernatant and wash pellet with 10 mL 1X PBS. Combine identical samples if necessary, then repeat step 2.
- Aspirate supernatant and gently resuspend pellets in 5 mL of 10 mM TRIS.Cl, pH 9.0.
- Transfer cell suspension to the Dounce homogenizer and incubate on ice 10 minutes.
All steps below this line should be performed at 4°C
- Dounce each aliquot 25 times with the tight pestle (A)
- Centrifuge the suspension in a 15 mL Falcon tube at 900g x 5 minutes to pellet nuclei.
- Pipet supernatant into a 15 mL Falcon tube and save it on ice.
- Resuspend pellet in 2 mL of 10 mM TRIS.CL, pH 9.0 and Dounce again.
- Centrifuge the suspension in a 15 mL Falcon tube at 900g x 5 minutes to pellet nuclei.
- Combine this supernatant with the initial reserved supernatant. Supernatant should appear translucent white.
- Layer the ~7 mL of homogenized virus supernatant onto 6.0 mL cushion of 36% sucrose in 10 mM TRIS.CL, pH 9.0 in Beckman Ultra-Clear centrifuge tubes.
- Centrifuge at 18K g, 80 minutes at 4°C.
- Aspirate the supernatant and sucrose, being careful not to disrupt the pellet.
- Resuspend pellet in 1.0 mL of 1 mM TRIS.CL, pH 9.0 (This volume is calculated for 10 plates, adjust as necessary)
- Aliquot and store at -80°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.
*'''~~~~''': There will occasionally be a viscous, fluffy white layer over the pellet (nuclear DNA?). This doesn't appear to impact the resulting titers, but adequate, homogenous resuspension in this case is critical.