https://eln.excellgen.com/index.php?action=history&feed=atom&title=Baculovirus_Generation_and_Amplificaiton 2026-10-18T17:25:28Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=Baculovirus_Generation_and_Amplificaiton&diff=3891&oldid=prev 2012-04-16T16:55:07Z

<p>Created page with &quot;== Generation of Baculovirus == 1. Subclone gene into pFastBac-DUAL&lt;br&gt;2. Select and isolate the recombinant donor plasmid.&lt;br&gt;3. Transform the recombinant donor plasmid into DH...&quot;</p> <p><b>New page</b></p><div>== Generation of Baculovirus ==<br /> <br /> 1. Subclone gene into pFastBac-DUAL&lt;br&gt;2. Select and isolate the recombinant donor plasmid.&lt;br&gt;3. Transform the recombinant donor plasmid into DH10Bac E. coli and select the recombinant bacmid.&lt;br&gt;4. Seed 9 × 105 Sf-9 cells in 2 mL insect cell medium to each 35-mm dish.&lt;br&gt;5. Transfect the recombinant bacmid into Sf-9 cells&lt;br&gt;6. After transfection, wash the Sf-9 cells with 2 mL medium and continue to culture the cells at 27 °C.&lt;br&gt;7. Harvest the recombinant virus in the culture medium at 72 h posttransfection. Determine the virus titer<br /> <br /> == Amplification of Baculovirus&lt;br&gt; ==<br /> <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.&lt;br&gt;2. Remove the medium and inoculate the virus at a low MOI. Replenish complete medium to a final volume of 0.5–1 mL.&lt;br&gt;3. Gently shake the flask on a rocking plate at room temperature for 1 h.&lt;br&gt;4. Add complete medium to a final volume of 5–7 mL. Incubate in a 27 °C humidified incubator.&lt;br&gt;5. Collect the medium at 72–96 h postinfection and centrifuge at 1,000 × g for 5 min.&lt;br&gt;6. Collect and store the virus supernatant at 4 °C, protected from light. Store at –80 °C for long-term storage.&lt;br&gt;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.&lt;br&gt;8. If concentrated baculovirus is required, load 33 mL virus supernatant in each of six 38 mL ultracentrifuge tubes.&lt;br&gt;9. Underlay with 3 mL sucrose cushion solution to each tube. &lt;br&gt;10. Centrifuge at 80,000 × g (24,000 rpm in an SW28 rotor) for 75–90 min at 4 °C.&lt;br&gt;11. Decant the supernatant and carefully remove traces of sucrose. The viral pellet appears translucent white.&lt;br&gt;12. Resuspend the pellets in 1–2 mL PBS (pH 6.2).&lt;br&gt;13. Filter the concentrated virus through 0.22 μm filters and determine the virus titer. Store at 4 °C.&lt;br&gt;<br /> <br /> &lt;br&gt;<br /> <br /> [[Category:Protocols]]</div>

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