AB Vector - Harvest troubleshooting

  Troubleshooting in generation of P1 baculovirus stocks
  If the monolayer reached 100% confluency and then overgrown, and the cells do not look infected, then something went wrong and the virus titer is low. Among probable reasons is touching the walls of reservoir at addition of liquids at transfection or too dense initial monolayer, which overgrew faster than the virus could infect majority of the cells. Do not get discouraged. Harvest the monolayer and the cell culture liquid and seed into 2-3 80 cm2 vent/close caps T-flasks. Into each flask add fresh medium up to 17 ml and continue the incubation for another several days, until the signs of infection become pronounced and about 50% of the cells collapse. Unite the cell culture liquids into a 50 ml conical tube, centrifuge at 1,500 r.p.m. for 15 min in a swing rotor table top centrifuge and collect the supernatant. Store P1 virus stock at 4oC.