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 titers. 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.