flashBAC Manual - Oxford Expression Technologies

8 Production of recombinant baculoviruses usingthe flashBAC systemOverviewProtocol 8.1 describes the method for making recombinantviruses in 35 mm dishes and protocol 8.4 gives guidance onadapting this method for use in automated or robotic systems.The following procedures must be carried out using aseptictechnique, as the DNA/liposome complexes will be introducedinto insect cells maintained in antibiotic-free medium.8.1 Production of recombinant baculovirus by cotransfectionof insect cells with flashBAC DNAand a transfer vector (containing the gene underinvestigation)Provided:• flashBAC DNA(use 100 ng [5 µl] per co-transfection [20 ng/µl]).• Positive-control transfer vector DNA(use 500 ng [5 µl] per co-transfection [100 ng/µl).Required:• 35 mm tissue culture treated dishes seeded with insectcells in a sub-confluent monolayer (Sf9 or Sf21) [seesection 10 for information about insect cells].• Serum-free insect cell culture medium.If using serum supplemented medium, you will needmedium with and without 10% foetal bovine serum.OXFORD EXPRESSION TECHNOLOGIES 20