Lentivirus harvest and concentration


Posted by xwang
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Protocol
Materials: 
  • Beckman Ultracentrifuge tubes (1 X 3.5 " or 9/16 " X  3 ½" )
  • Beckman Ultracentrifuge fitted with a SW-28 or SW-41 rotor
  • Serum Free DMEM
  • PBS
  • 10ml syringe
  • 0.45 μm syringe filter tips
  • 10% bleach
Methods: 
  1. Harvest virus with a 0.45um filter tip with a 10ml syringe, into a Beckman ultracentrifuge tube
  2. Bleach all virus producing cells, culture plates, syringes and filters with 10% bleach and allowed to stand for 45 minutes before discarding
  3. Balance all virus containing tubes with serum free DMEM (to 0.1g)
  4. Spin tubes at 25000rmp or 24000rmp for 90 mins in 4 degrees in a SW-28 or SW-41 rotor
  5. Discard supernatant into 10% bleach containing vessel
  6. Blot excess liquid with kimwipes. Turn tubes upside down in a rack and allow to drain for one minute
  7. Resuspend viral pellet in 100ul of PBS. Let virus sit overnight in 4 degrees
  8. Bleach all empty tubes with 10% bleach for45 minutes before discarding