Brennan:Partial purification of vaccinia virus

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

1. Scrape infected cells from dish, using a rubber policeman if necessary, and combine the cells in 50 mL tubes.
2. Pellet cells at 900g x 10 minutes.
3. Aspirate supernatant and wash pellet with 10 mL 1X PBS. Combine identical samples if necessary, then repeat step 2.
4. Aspirate supernatant and gently resuspend pellets in 5 mL of 10 mM TRIS.Cl, pH 9.0.
5. Transfer cell suspension to the Dounce homogenizer and incubate on ice 10 minutes.

All steps below this line should be performed at 4°C

1. Dounce each aliquot 25 times with the tight pestle (A)
2. Centrifuge the suspension in a 15 mL Falcon tube at 900g x 5 minutes to pellet nuclei.
3. Pipet supernatant into a 15 mL Falcon tube and save it on ice.
4. Resuspend pellet in 2 mL of 10 mM TRIS.CL, pH 9.0 and Dounce again.
5. Centrifuge the suspension in a 15 mL Falcon tube at 900g x 5 minutes to pellet nuclei.
6. Combine this supernatant with the initial reserved supernatant. Supernatant should appear translucent white.
7. 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.
8. Centrifuge at 18K g, 80 minutes at 4°C.
9. Aspirate the supernatant and sucrose, being careful not to disrupt the pellet.
10. Resuspend pellet in 1.0 mL of 1 mM TRIS.CL, pH 9.0 (This volume is calculated for 10 plates, adjust as necessary)
11. Aliquot and store at -80°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.

*'''~~~~''': 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.