Lentivirus concentration using ultracentrifugation Neville Sanjana ([email protected]) Zhang Lab, August 2012 Harvest lentivirus at 48-60 hours after media replacement/transfection. Lentivirus is stable at 4C. Work quickly during all room temperature steps. 1. Spin viral harvest in Beckman GS-6R centrifuge for 10 min @ 3000 rpm at 4C to pellet any cell debris. During this spin, prepare the following: • Prepare konikal tubes, with rubber round bottom tube adaptors in ultracentrifuge tubes. Max volume is 33mL, use a minimum of 30mL, adding media if necessary. • Prepare ultracentrifuge rotor. 2. Filter supernatant through 0.45um Steriflip filter (HV/PVDF low protein binding membrane). 3. Pour filtered supernatant into konikal tubes and spin for 2 hrs at 24,000 rpm at 4C using SW32Ti or SW28Ti. 4. Carefully pour off supernatant and place inverted konikal tube on a paper towel to remove all remaining media. • Supernatant contains trace infectious particles. Dispose of biohazard waste properly. 5. Resuspend viral pellet using 200-ul pipette tip with desired volume of DMEM supplemented with 1% BSA to concentrate virus 100x or more. 6. Place ultracentrifuge tube within a tightly sealed 50ml Falcon tube and allow resuspension overnight at 4C for the pellet to completely dissolve. 7. Next morning: Aliquot virus and store aliquots at -80C. Thaw an aliquot on ice before each use; do not refreeze.

Materials: Konikal tubes : Beckman 358126 Tube adaptors : Beckman 358156