adapted from Cell Biology, A Lab Manual, second edition, volume 1 -Grow up P11 cells in 15 cm plates to 70 – 80% confluence. -Infect cells with a MOI (multiplicity of infection) of 1 – 10 PFU per cell (or enough virus such that within 48 hours cells are mostly rounded up, but not complete detached from the plate. -When cytopathic effect is nearly complete (i.e. most cells rounded but not yet detached from the dish), harvest media and cells by scraping off the dish. Centrifuge in 250 ml disposable bottles (pooling 8 plates of similarly infected cells per bottle) at 800rpm for 15 min. Discard media (pour off and bleach) and resuspend cell pellet carefully in 10 ml 0.1 M Tris-HCL, pH 8.0,-transfer to 15 ml conical tube. At this point pellets can be stored at –70° until used or re-spin down and resuspend cell pellet in 7.0 ml 0.1 M Tris-HCL, pH8.0. The cell pellet + 7.0 mls should give a final volume of approximately 7.5 ml total. (The 7.5 ml/8 plates was figured to yield approximately 13 ml of prep to go into a 12.5 ml quickseal tube prior to CsCl banding below). -Add 0.1 vol 5% Na deoxycholate (750 ml). Mix well and incubate at room temperature for 30 minutes. This disrupts the cells without disrupting the virions, resulting in a highly viscous suspension. -Add 0.01 vol 2 M MgCl2 (82.5 ml) and 0.005 vol Dnase I solution (42 ml), then mix well. Incubate at 37° for 30 – 60 minutes, mixing every 10 minutes. The viscosity should be reduced at this point. -Add 1.8 ml Cs Cl solution for each 3.1 ml virus suspension. Be sure that the saturated CsCl solution is equilibrated to room temperature, as this affects the concentration. (if you have been going by volumes above, should be total of approxmiately 8.3 ml to which you add 4.8 ml CsCl solution). -Transfer virus to quickseal tubes (approximately 12.5 mls fills each) and spin in Beckman 70Ti for 16 – 20 hr at 4° and 35K rpm. -Collect the viral bands and pool (collect by puncturing the top of the tube with a hot needle, then puncturing the bottom and collecting band as it flows out of the tube). -Centrifuge pooled virus in the Beckman SW rotor for 16 – 20 hr at 4° and 35K rpm. Overlay virus with mineral oil prior to balancing as these tubes are not sealed. -Collect the virus (I pull band with a needle at this point) in smallest volume possible, transfer to a Slide-A-Lyzer dialysis cassette, and dialyze in cold room against three changes of 500 vol 10 mM Tris-HCl, pH 8.0 for at least 4 hr each change. -After dialysis, add sterile glycerol to a final concentration of 10% glycerol. Store the purified virus at –70° in small aliquots. For solutions: (and try to keep everything sterile) Sterile filter 5% Na Deoxycholate and 2 M MgCl2 Autoclave 0.1 M Tris-HCl, pH 8.0 Dnase I solution: Dissolve 100 mg bovine pancreatic deoxyribonuclease I (Dnase I) in 10 ml of 10 mM Tris-HCL, pH 7.4, 50 mM Na Cl, 1 mM DTT, 0.1 mg/ml BSA and 50% glycerol. Store in small aliquots at –20° Use sterile TE for saturated CsCl solution and store at 4°, but equilibrate to RT prior to use. Prepare sterile glycerol by autoclaving.
