Most available baculovirus vectors are constructed on a plasmid basis and provide ampicillin resistance.

Operation method

basic program

Materials and Instruments

Insect Cells
Fetal Bovine Serum Liposome Transfection Agents
Petri dishes Conical flasks Incubators Centrifuges Rotors

Move

1. Inoculate ^2x106 Sf9 cells in 60 mm dishes in complete or serum-free medium containing 10% fetal bovine serum and incubate at 27°C for 30-60 min to allow the cells to adhere to the walls.

2. According to each culture dish to be transfected, draw 40 ul of sterile water into a sterile polystyrene tube, add 5 ul of linearized AcMNPV DNA and 5 ul of plasmid DNA at 100 ng/ul. Mix Lipofectin well, add 50 ul of it into the DNA solution, mix gently and incubate at room temperature for 15 min.
3. During the 15 min incubation period, replace the culture medium in the Petri dish with 1.5 ml of complete culture medium without fetal bovine serum. 4.

4. Add the Lipofectin-DNA complex dropwise to the Petri dish while gently rotating the dish to mix. Incubate at 27℃ for 4~5 h.5. Add 1.5 ml of complete culture medium containing 10% fetal bovine serum (or serum-free culture medium containing 5% fetal bovine serum) to each petri dish, and incubate at 27℃ in a humidified incubator for 60-72 hours.6. Transfer the transfection supernatant (consisting of culture medium and virus) from each fertile dish to a sterile 15 ml conical centrifuge tube and centrifuge at 4℃ for 10 min at 1,000 g. Transfer the viral supernatant to a new sterilized tube and store at 4℃ away from light.

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