Amplification of baculovirus

Amplification of baculovirus
The idea is to infect the cells and wait for multiple rounds of infection to occur over a 5
day period. This will lead to release of more virus due to cell lysis and therefore higher
1. Start with a T25 flask containing 60-70 % confluent Sf21 (or Sf9) cells.
2. Aspirate the medium. Add 0.5-1 ml of virus and then complete medium up to 1-2
ml total volume (whatever is the smallest volume JUST sufficient to cover the
cells on a rocking table). Infect for 1 hr at room temperature on a rocking table –
don’t let it rock too fast or the cells will come off.
3. Aspirate virus and replace with fresh medium (about 5 ml).
4. Harvest virus stock at 5 days post infection. Spin down in a tabletop centrifuge at
1200 rpm for 5 min. Transfer the supernatant to a new tube and save it at 4 ˚C as a
virus stock. Wrap the tube with metal foil to protect from light. For a typical
infection of a p60 or p100 dish I use 0.5-1 ml of virus stock and look at protein
expression 48 hrs post infection.
5. To amplify greater volumes, go on to use larger flasks (T75, or T150) and scale
up everything accordingly.