Editing Adenovirus AdEasy (section)

== Preparation of High Titer Viral Stocks  ==

1. Plate 911 or 293 cells in T-75 flasks to be 90% confluent at time of infection (about 1x107 cells/T-75). Usually, fiften to twenty T-75 flasks are sufficient to make a high titer stock. 

2. Infect cells with virus sup at a multiplicity of infection (MOI) of 5 to 10 PFU per cell. When all cells have rounded up and about half of the cells are detatched (usually at 3 to 4 days post infection), harvest and combine all flasks. Spin 5 min in a benchtop centrifuge (~500 g), and remove the supernatant. 

3. Resupend pellet in 8.0 ml sterile PBS. Perform four cycles of freeze/thaw/vortex. Centrifuge lysate in Sorvall HS4 rotor at 6000 rpm (7000 g) 4oC for 5 min. 

4. Weigh 4.4 grams of CsCl in a 50-ml conical tube, transfer 8.0 mls of clear virus sup to the tube, and mix well by vortexing. Transfer the CsCl solution (about 10 ml, density of 1.35 g/ml) to a 12 ml polyallomer tube for SW41 rotor. Overlay with 2 ml mineral oil. Prepare a balance tube. Spin the gradient in SW41 rotor at 32,000 rpm, 10oC, for 18 to 24 hours. 

5. Collect virus fraction (about 0.5 to 1.0 ml) with a 3cc syringe and an 18g needle. Mix with equal volume 2X Storage Buffer (2X Storage Buffer = 10 mM Tris, pH 8.0, 100 mM NaCl, 0.1% BSA, and 50% glycerol, filter sterilized). Store virus stocks at -20oC. 

6. Check viral titer by GFP (preferred) of by plaque assays (see below) or by immunohistochemical staining, or simply read OD at 260 nm. To read OD, add 15 µl virus to 15 µl blank solution (Blank Solution = 1.35g/ml CsCl mixed with equal vol 2X Storage Buffer) plus 100 µl TE/0.1% SDS; vortex 30 seconds, centrifuge 5 min. measure A260. One A260 unit contains ~1012 viral particles (particles:infectious particles =~20:1). 

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