Editing Adenovirus Invitrogen (section)

== Adenovirus Amplification Procedure  ==

Follow the procedure below to amplify your adenoviral stock using 293A cells. Make sure that your 293A cells are healthy at the time of plating. 

1. The day before infection, trypsinize and count the 293A cells, plating them at 3 x 106 cells per 10 cm plate. Plate cells in 10 ml of normal growth medium containing serum. 

2. On the day of infection, verify that the cells are at 80-90% confluency before proceeding. Add the desired amount of crude adenoviral stock (e.g. 100 ÎŒl) to the cells. Swirl the plate gently to mix. 

3. Incubate the cells at 37°C in a CO<sub>2</sub> incubator and allow infection to proceed until 80-90% of the cells have rounded up and are floating or lightly attached to the tissue culture dish (typically 2-3 days post-infection). This indicates that cells are loaded with adenoviral particles. 

Note: If you have used less than 100 ÎŒl of crude viral stock or a lower titer stock for infection, you may need to perform a longer incubation. 

4. Harvest adenovirus-containing cells by squirting cells off the plate with a 10 ml tissue culture pipette. Transfer cells and media to a sterile, 15 ml, capped tube. 

5. Place the tube containing harvested cells and at -80°C for 30 minutes. Remove tube and place in a 37°C water bath for 15 minutes to thaw. Repeat the freezing and thawing steps twice. 

6. Centrifuge the cell lysate in a table-top centrifuge at 3000 rpm for 15 minutes at room temperature to pellet the cell debris. 

7. Transfer the supernatant containing viral particles to cryovials in 1 ml aliquots. Store the viral stocks at -80°C.