Author
Date
Version
SOP #
Dan McManus
September 2007
1
V002
Objective: This SOP addresses the purification of vesiculoviruses following their isolation
from infected cell monolayers using protocol SOP-V001
Required reageants/equipment:
1x PBS
15% glucose (0.22um filter sterilized, prepared in PBS)
20% sucrose (0.22um filter sterilized, prepared in dH2O
Ultraclear open top tube (Beckman # 344059)
SOP-V002: Sucrose Cusion Purification of Vesiculoviruses
1. Remove all medium and perform two rinses of the centrifuge tubes with 10ml
sterile 1 x PBS, being careful not to disturb the viral pellet.
2. Resuspend viral pellet with the appropriate volume of PBS (e.g. 10ml if using
SW41 open top tubes).
3. Add 1.1ml of 20% sucrose to an ultraclear open top tube, and gently layer the
10ml virus on top of the sucrose layer.
4. Add tubes to SW41 adapters and screw on the caps. Weigh and balance by
adding PBS to the appropriate balance tube. Run the rotor with all 6 balanced
adapters in place (this is important).
5. Spin at 125,000g (26,900RPM) at 4°C for 90 minutes using max
acceleration/deceleration.
6. Carefully remove tubes into the tissue culture hood. Carefully discard the
supernatant with a pipette, being careful not to disturb the viral pellet at the
bottom of the tube.
7. Resuspend the viral pellet in the appropriate volume of 15% glucose.
8. Once the viral pellet is completely resuspended, aliquot and store at -80°C for
subsequent titration.
This protocol is shared by the Stojdl Lab under a creative commons license.