Editing Adenovirus Invitrogen (section)

== Preparing a Crude Viral Lysate  ==

After you have harvested adenovirus-containing cells and media, you will use several freeze/thaw cycles followed by centrifugation to prepare a crude viral lysate. The freeze/thaw cycles cause the cells to lyse and allow release of intracellular viral particles. 

1. Place the tube containing harvested cells and media 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.<br>Note: Do not incubate samples at 37°C for longer than 15 minutes. 

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

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

The titer of the initial viral stock obtained from transfecting 293A cells generally ranges from 1 x 10<sup>7</sup> to 1 x 10<sup><sub>8</sub></sup> plaque-forming units (pfu)/ml.<br>When stored properly, viral stocks of an appropriate titer should be suitable for use for up to one year.