Generation of a high-titer viral stock from a recombinant adenovirus. Plaque purification prior to amplification is highly recommened to insure the purity and stability of the recombinant adenovirus. Amplified adenoviruses are purified by CsCl banding (ultracentrifugation)
Scale: 1 L AdExpress-293A culture
Time Required: 2-6 weeks
Includes:
- Amplification of a recombinant adenovirus in AdExpress-293A cells at the desired volume.
- Harvest adenovirus. Three freeze/thaw cycles are performed and cell debris is removed by centrifugation.
- Purification on a CsCl step gradient followed by a continuous CsCl gradient using an ultracentrifuge.
- Desalting and elute virus using a storage buffer containing glycerol
- Make viral stock aliquots.
Deliverable: Purified adenovirus. The final titer is variable and depends on the nature of the recombinant adenovirus to be amplified. On average 2.5x10e13 particles are obtainined. For an accurate infectious titer, a TCID50 or plaque assay can be performed on the final product.
