Adenovirus Production Outline

Steps Performed By	Adenovirus Production Outline
Investigator	Obtains pShuttle plasmid from the Gene Vector Core Clones gene of interest into pShuttle Transforms plasmid and selects for Kanamycin-resistant colonies Screens mini-prep DNA from colonies by restriction digests Sends at least 100ug of the plasmid to the Gene Vector Core
Gene Vector Core	Expression cassette excised from pShuttle Expression cassette ligated into the Adeno Viral DNA Ligation reaction digested with Swa I and transformed into Stbl2 cells Ampicillin-resistant colonies screened by restriction digests Maxi-prep of the final plasmid are made Adenoviral DNA digested with Pac I HEK 293 cells transfected and monitored for plaque formation Plaques picked and small-scale expansion performed
Investigator	Positive samples sent to investigators to test and select sample for final expansion & purification
Gene Vector Core	Medium-scale expansion of investigator's selected sample for production of lysate Viral lysate used to infect cell factory Large-scale expansion harvested and purified on two CsCl gradients Smaple desalted, aliquoted and frozen QA/QC testing Virus shipped by the Gene Vector Core to the investigator

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