Editing Adenovirus Purification Clontech (section)

== Test the Titer of the Adenoviral Stock  ==

<br>1. Determine the optimal amount of viral stock needed to infect 293 cells. To do this, test the viral titer in a small scale cytopathic effect assay, using culture conditions that closely approximate those that will be used for the actual adenoviral prep. 

a. Seed low passage HEK 293 cells on a 12-well tissue culture dish at a density of 1x105 cells/cm2, using 0.28 ml/cm2 medium. At 1 ml/well, this is equivalent to ~3.57x10^5 cells/well. 

b. Place the cells at 37oC and 5% CO2 while you dilute the adenovirus. 

c. Infect the cells with a range of adenoviral concentrations. If the titer of adenovirus stock is known, aim for a multiplicity of infection (M.O.I.) of 1-2. if the titer is unknown, infect the cells with a range of dilutions, starting with 1 ml of stock and serially diluting 3X from there. 

d. Incubate the cells at 37oC and 5% CO2 until the cytopathic effect is complete; this should take approximately 3-5 days. 

Note: The cytopathic effect (CPE) refers to the morphological changes that the cells undergo after infection. Infected cells typically remain intact, but round up and may detach from the dish individually or in âgrape-likeâ clusters that float in the medium. Optimally, the purification assay should be performed when 50% of the cells are detached, and the remainder are attached but rounded. 

2. Use the results from the test titer to determine the optimal amount of virus to use in the actual purification prep. The full scale prep requires seeding several 150 mm cell culture dishes with 1.46x10^7 cells/dish. Therefore, if 0.1 ml of adenoviral stock optimally infected the 3.57x10^5 cells used to seed the test titer, 4 ml of virus is needed for each 150 mm dish in the actual prep.