Editing Adenovirus Production (section)

== Mid Scale Amplification ==

1. Seed 3-5 x 10<sup>6</sup> cells in a T75 flask one day before infection.

2. Add 100 ul of the above IVS to the culture. Usually this will give a multiplicity of >0.5 PFU (plaque forming units). This amount of viruses will result in cytopathic effects (CPEs) within 48 hrs.

3. During 24 - 48 hr infection, examine the monolayer twice per day under the microscope for CPE. When CPE is nearly complete (i.e. most cells '''rounded''' '''but not yet detached from the flask'''), harvest cells by pipetting media up and down to wash the infected cells from the flask into the media.

<br/>4. Pool infected cells and medium. Pellet cells by centrifugation at 1000 g for 5 minutes. Remove supernatant, resuspend cell pellet in medium or in PBS or 10 mM Tris, pH 8.0, 100 mM NaCl. (0.25-0.5 mL per T75 flask)

5. Release the adenoviruses from the cell suspension with 4 freeze/thaw cycles. Centrifuge at 3000xg for 10 minutes to pellet the cell debris. Discard the pellet, transfer the supernatant to a clean tube, add 80% glycerol to a final concentration of 10% glycerol, make 250 ul aliquots, and store the&nbsp; Viral Stock (VS)&nbsp; at -80 °C.