Baculovirus Amplification (Pass 2 or Small Scale Pass 3)

  1. Remove 4x107 cells to a 50ml Falcon tube (~20ml at 2x106)

  2. Pellet at 1500rpm 5min. Aspirate supernatant.

  3. Resuspend in 0.5ml virus (at1x108) with 3.5ml fresh medium. MOI=0.5-1.0

  4. Incubate 60min at 280C with occasional swirling

  5. Add 36ml medium, mix and plate 20ml each on T-150 flasks

  6. Incubate 3-4 days at 280C

  7. Harvest supernatant and spin down cells and debris.



Baculovirus Amplification (Pass 3 or Large Scale)


  1. Divide 150ml of cells at 2x106 (3x108 total) into three 50ml Falcon tubes.

  2. Pellet cells at 1000rpm for 8min. Aspirate supernatant.

  3. Resuspend in Pass 2 virus at MOI of 0.5 ~1.5ml at 1x108 for all the three tubes

  4. Add 9x virus volume of fresh medium (~13.5ml)

  5. Incubate at 280C for 1h with occasional swirling

  6. Transfer mix to fresh flask with 300ml of fresh medium

  7. Incubate 3-4days at 280C

  8. Harvest to six 50ml Falcon tubes

  9. Spin 10min at 1000rpm.Store at 40C.

Note: If the supernatant is cloudy check by microscopy for contamination.