https://eln.excellgen.com/index.php?action=history&feed=atom&title=Adenovirus_Prestock 2026-10-18T02:15:52Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=Adenovirus_Prestock&diff=3333&oldid=prev 2012-02-20T04:10:09Z

<p></p> <p><b>New page</b></p><div>1. 6 to 16 hours before transfection, plate 10^6 293A cells in several 25-cm2 tissue culture flask(s) with DMEM + 10% FBS. Plate at least two flasks. One plate may work as control plate and help to follow vector amplification. <br /> <br /> 2. Digest 1 ug of recombinant pAd5B (backbone) adenoviral plasmid and pAd5 plasmid (with GOI) with 30 U of PacI to separate adenovirus genome from bacterial sequences. <br /> <br /> To ensure complete digestion, 6 h later add another 30 U of PacI. Digest overnight. <br /> <br /> 3. Precipitate digested DNA with two volumes of ethanol and resuspend in 20 mL of sterile ddH2O. <br /> <br /> 4. Perform a standard transfection using 2 ug of PacI digested plasmids&amp;nbsp; per 10^6 HEK-293 cells. <br /> <br /> 5. Incubate the DNA with GeneExpressoMax transfection reagent for 20 min at room temperature. <br /> <br /> 6. Add DNA complexes dropwise to the cells. Incubate at 37°C and 5% CO2 for 6 days. <br /> <br /> 7. Scrape/harvest the medium and cells. Freeze/thaw three times to release adenovirus from cells. <br /> <br /> 8. Centrifuge at 4 °C for 10 min and 500 x g and discard the pellet. <br /> <br /> 9. Use all the centrifuged crude lysate to infect 8 × 10^6 HEK-293 cells (at 70–80% confluency) in a 10-cm plate. <br /> <br /> 10. Incubate at 37°C and 5% CO2 until general cytopathic effect is observed (usually between 3 and 6 days). <br /> <br /> 11. Harvest medium and cells. Freeze/thaw three times to release adenovirus from cells. <br /> <br /> 12. Centrifuge at 4°C for 10 min and 500 × g. Discard the pellet and keep the supernatant. <br /> <br /> 13. Use all the previous centrifuged crude lysate to infect 4 × 10^8 HEK-293 cells (at 70–80% confluency) in twenty 25-cm plate. <br /> <br /> 14. Incubate at 37°C and 5% CO2 until general cytopathic effect is observed (usually between 32 and 38 h). <br /> <br /> 15. Harvest medium and cells. Freeze/thaw three times to release adenovirus from the cells. <br /> <br /> 16. Centrifuge at 4°C for 10 min and 500 × g. Discard the pellet and keep the supernatant (viral-prestock). <br /> <br /> 17. Titer the viral pre-stock by anti-hexon staining method. <br /> <br /> 18. Aliquot and store viral pre-stock at −80°C.&lt;br&gt; <br /> <br /> [[Category:Protocols]]</div>

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