Author
Date
Version
SOP #
Kang Hu
October 2005
1
V003
Objective: This SOP addresses the generation of Vaccinia Virus stocks
Required reageants/equipment:
VV (vTF7-3) seed stocks
U2OS cell medium
1mM Tris pH 9.0
36% sucrose (made up in 1mM Tris pH 9.0)
1 x Trypsin/EDTA
SOP-V003: Generation of Purified Vaccinia Virus Stocks
Monolayer Infection and Virus Isolation
1. Prepare ten 150mm dishes of confluent U2OS cells.
2. Infect with VV seed stock at a MOI of 0.1 in 750ul. Allow virus to adsorb for 45 mins.,
then add 15ml fresh medium.
3. Wait ~ 3d until extensive cytopathic effect is visible. Collect cells into 4 x 50ml falcon
tubes.
4. Centrifuge at 2000g for 10 mins. Discard supernatant. Resuspend cell pellet in 20ml
1mM Tris pH 9.0 per falcon tube.
5. Freeze/thaw cell pellet three times (place at -80°C then +37°C), vortexing vigorously
inbetween temperature extremes.
6. Centrifuge at 2000g for 10 mins. Save the supernatant, which contains the virus.
7. Wash the pellet by adding 20ml 1mM Tris pH 9.0 and vortexing.
8. Centrifuge at 2000g for 10 mins. Pool the supernatant and discard the pellet.
Sucrose Purification
9. Add 10ml 36% sucrose into two Oak Ridge-type tubes.
10. Carefully pipette 20ml of virus supernatant on top of the sucrose, being careful not to
disturb the interface.
11. Centrifuge at 11,500RPM (20,700g) in a swinging bucket JS13.1 rotor for 90 mins. at
4°C.
12. Immediately aspirate supernatant, being careful not to disturb the viral pellet.
13. Resuspend pellet in 1ml 1mM Tris pH 9.0 and pool.
14. Aliquot into 200ul volumes and store at -80°C.
This protocol is shared by the Stojdl Lab under a creative commons license.