Lentiviral Harvest and Concentration


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Protocol
Methods: 

Lentiviral Harvest


___ Virus harvested from a 10cm or 15cm plate with a 0.45 μm filter tip, using a 10ml
or 30ml syringe into an ultraclear Beckman centrifuge tube ( 1 X 3.5 " or 9/16 "
X 3 ½" ).
___ All virus producing cells, culture plates, syringes, filters bleached with 10% bleach
and allowed to stand 45 min before discarding.
Note: Filtered viral supernants may be used directly if a high titer is not necessary.
Concentration may be necessary for use with ES cells, injection or other difficult to infect
cell lines. Concentration of lentivirus by ultracentrifugation is possible only for
lentviruses which have a VSV-G coat. Ecotropic coats are unable to withstand the g
force of ultricentrifugation and must be concentrated by a different method.


Lentiviral Concentration by Ultracentrifugation
___ All virus containing tubes balanced with serum free DMEM and an APX-402 scale.
___ Virus centrifuged @ 25,000rpm for 90 min. @ 4° C in SW-41 or SW-28rotor.
___ Supernant discarded into a vessel containing 10% bleach. Excess liquid absorbed
with kimwipe and tubes inverted for 2-3 minutes to drain.
___ Viral pellet resuspended in 100-150ul of PBS. Virus allowed to sit @ 4° C
overnight.
___ All empty tubes bleached with 10% bleach for 45 min before discarding