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... Ordinarily, a vector stock is obtained simply by pipetting off the medium from an infected culture. The titer of the virus in the culture medium is sufficient for most purposes. For those who want a more efficient infection, there are two simple alternatives: either the virus in the medium can be concentrated (usually by sedimentation in an ultracentrifuge), or infection can be carried out by cocultivating the target cells and the virus-producing cells. Each protocol has particular advantages and disadvantages.

Cocultivation is simple and effective; however, the producer cells usually need to be removed from the culture at some point. If the target cells express a selectable marker, the cells can be separated using the appropriate selection procedure. Alternatively, the virus-producing cells can be treated with mitomycin C before the target cells are added. Mitomycin C treatment “kills” the cells (they cannot be passaged), but they remain metabolically active and continue to produce virus with only a modest reduction in titer.

Concentrating the virus by sedimentation also works, but the recovery of infectious virus is not always complete. Quite often, the physical concentration is higher than the concentration of the viral titer, probably due primarily to the fact that the viral pellet is difficult to resuspend. Some people prefer to sediment the virus onto a dense sucrose cushion rather than the bottom of a tube. In any case, do not subject the virus to significantly more sedimentation than what is needed to pellet the virions. When resuspending the concentrated virus, be patient and gentle. Let the pellet sit on ice, then resuspend it by gentle pipetting (a pipetteman works well) rather than using prolonged or vigorous vortexing.




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