Editing Adenovirus Production (section)

== 1st Round Amplification ==

1.&nbsp;Wash cells with PBS, Use trypsin to digest cells, add 5 ml PBS.&nbsp;Transfer to a clean tube.

2. Spin down cells at max speed (microcentrifuge) for 30 sec, remove PBS and add 0.2 ml fresh PBS.

3. Release viruses from cells by three freeze/thaw cycles ( in 37 oC incubator and dry ice).

4. Add the entire lysate to 10 cm plate or T75 flask with 293A cells (50%) conflucence.

5. When cells are round,&nbsp; (cytopathic effects, CPEs), harvest adenovirus-containing cells by squirting cells off the plate with a 5 or 10 mL sterile serological pipette. Transfer cells and media to a sterile 15 ml tube. Scraping the cells into the medium with a cell lifter, if necessary.

6. Spin down cells, transfer medium to a clean tube, which can be used for futher amplification.&nbsp;&nbsp; Add 0.5 ml PBS to cell pellet.

7. Release viruses from cells by three freeze/thaw cycles (in 37 oC mixer/incubator and dry ice).

8. Centrifuge the cell lysate in a eppendorf centrifuge at 13000 rpm for 2 minutes at room temperature to pellet the cell debris.

9.&nbsp; Transfer the supernatant to a clean tube, add 80% glycerol to a final concentration of 10% glycerol,&nbsp; make 250 ul aliquots, and store the Crude Viral Lysate (Initial Viral Stock, IVS) at -80 °C.