Editing Baculovirus Generation and Amplificaiton

== Generation of Baculovirus ==

1. Subclone gene into pFastBac-DUAL<br>2. Select and isolate the recombinant donor plasmid.<br>3. Transform the recombinant donor plasmid into DH10Bac E. coli and select the recombinant bacmid.<br>4. Seed 9 × 105 Sf-9 cells in 2 mL insect cell medium to each 35-mm dish.<br>5. Transfect the recombinant bacmid into Sf-9 cells<br>6. After transfection, wash the Sf-9 cells with 2 mL medium and continue to culture the cells at 27 °C.<br>7. Harvest the recombinant virus in the culture medium at 72 h posttransfection. Determine the virus titer

== Amplification of Baculovirus<br> ==

1. Seed Sf-9 cells to T-25 flasks at ~50–70% confluency and allow the cells to attach at 27 °C for at least 1 h.<br>2. Remove the medium and inoculate the virus at a low MOI. Replenish complete medium to a final volume of 0.5–1 mL.<br>3. Gently shake the flask on a rocking plate at room temperature for 1 h.<br>4. Add complete medium to a final volume of 5–7 mL. Incubate in a 27 °C humidified incubator.<br>5. Collect the medium at 72–96 h postinfection and centrifuge at 1,000 × g for 5 min.<br>6. Collect and store the virus supernatant at 4 °C, protected from light. Store at –80 °C for long-term storage.<br>7. To amplify the virus stock to a larger scale, the process can be repeated by infecting the cells cultured in larger spinner flasks at a low MOI.<br>8. If concentrated baculovirus is required, load 33 mL virus supernatant in each of six 38 mL ultracentrifuge tubes.<br>9. Underlay with 3 mL sucrose cushion solution to each tube. <br>10. Centrifuge at 80,000 × g (24,000 rpm in an SW28 rotor) for 75–90 min at 4 °C.<br>11. Decant the supernatant and carefully remove traces of sucrose. The viral pellet appears translucent white.<br>12. Resuspend the pellets in 1–2 mL PBS (pH 6.2).<br>13. Filter the concentrated virus through 0.22 μm filters and determine the virus titer. Store at 4 °C.<br>

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[[Category:Protocols]]