flashBAC Manual - Oxford Expression Technologies

healthy cells from a log-phase culture [see section10] and to seed the cells at the correct cell density sothe resulting monolayer is even and sub-confluent.Use 1.5 x 10 6 Sf21 or 1 x 10 6 Sf9 cells/dish in a 2 mlvolume of medium.• Ensure that the cells are evenly distributed over thesurface of the dish and leave to settle at roomtemperature for 1 hour on a flat surface.2 During the 1 hour incubation period above, prepare theco-transfection mix of DNA and liposome reagent. Foreach co-transfection, pipette 1 ml serum-free,antibiotic-free medium into a sterile, disposablepolystyrene container (7 ml bijoux are convenient).3 Add an appropriate volume of transfection reagentas directed by the manufacturer and mix.• As a guide, use 5 µl Lipofectin ®transfection reagent.)(or alternative4 Next, add 100 ng flashBAC DNA (5 µl from the kit)and 500 ng transfer vector DNA (one with the geneunder investigation or the control provided in the kit [inwhich case use 5 µl]). Mix with gentle agitation orvortexing.• In the mock-transfection control, omit the DNA fromthe medium.5 Incubate at room temperature for 15 - 30 minutes toallow the liposome-DNA complexes to form.OXFORD EXPRESSION TECHNOLOGIES 22