Download Purification of Virus (Revision 1)

Adenovirus Purification (Chartier/Kuo)
Sample(s):
1. If harvested pellet/media was frozen, thaw at 37C.
2. Subject cell suspension to three freeze thaw cycles using liquid nitrogen and a 37C waterbath,
vortexing briefly after each cycle.
3. Centrifuge at 3000rpm and 4C for 10min using the Eppendorf Centrifuge 5810R.
4. During centrifugation, place 2.5ml of δ=1.4g/ml CsCl in PBS in four Beckman 9/16 x 3.75 in.
UltraClear Centrifuge Tubes. Carefully layer 2.5ml δ=1.25g/ml CsCl in PBS on top. Mark
interface with thin sharpie. (Assumes 20 T75s; can scale accordingly)
5. Following centrifugation, layer the virus containing supernatants on top of the CsCl gradients.
6. Balance tubes with PBS and centrifuge at 35k rpm and 4C for 60min using a SW40 rotor and
Beckman Coulter Optima L90k Centrifuge.
7. During centrifugation, place 10ml of δ=1.35g/ml CsCl in PBS in two Beckman 9/16 x 3.75 in.
UltraClear Centrifuge Tubes (one will be for virus, the other for balance).
8. Remove viral bands via sideband pull (upper band empty capsid; lower band infectious
particles)
a. Use a 3ml syringe with a 1.5inch 20 guage needle.
b. Insert needle just below the lower viral band. (Keep fingers out of the way in case
needle goes through the other side!)
c. Turn the open end of the needle up and remove material until the white virus band is
no longer visable.
d. Pour remaining liquid into waste container prior to removing needle.
e. Withdraw needle and layer material on CsCl cushion.
9. Add PBS to second tube to balance and centrifuge at 35k rpm and 4C overnight (16-20h)
using a SW40 rotor and Beckman Coulter Optima L90k Centrifuge.
10. Remove viral band via sideband pull (upper band empty capsid; lower band infectious
particles)
a. Use a 3ml syringe with a 1.5inch 20 guage needle.
Current as of 12-10-14
b. Insert needle just below the lower viral band. (Keep fingers out of the way in case
needle goes through the other side!)
c. Turn the open end of the needle up and remove material until the white virus band is
no longer visible.
d. Pour remaining liquid into waste container prior to removing needle.
e. Withdraw needle and transfer to 1.5ml tube.
11. Using Slide-a-lyzer system (Pierce), dialyze virus against dialysis buffer (3x 1L, 1hr each).
a. Can also dialyze against a different buffer (ie. PBS or Tris). Can also store virus in
CsCl
12. Aliquot dialyzed virus in 1.5ml tubes and stored at -80C in “
CsCl Solutions
1.25g/ml 36.16g CsCl + 100ml PBS
1.35g/ml 51.2g CsCl + 100ml PBS
1.40g/ml 62.0g CsCl + 100ml PBS
Filter Sterilize
Current as of 12-10-14
.”