SF9 Capsid Protocol
1) Split cells 1 confluent 100mm plate into 1 150mm plate.
Allow cells to recover for ~1 hr.
2) Infect 20 150mm plates of SF9 cells with 100ul of virus stock.
3) Incubate at 300C for 5 days.
4) Collect the cells by centrifugation and resuspend in 10ml PBS
plus complete (Roche protease inhibitor).
5) Dounce ~50 strokes on ice using the B pestal.
6) Spin at 8000 rpm/~10,000g in a Beckman SW41ti rotor for 15
min.
7) Resuspend the pellet in breaking buffer (15 ml).
8) Pass the sample through a Fluidizer 3 times.
9) Sonicate 30sec. x 3 with 1 min. between on ice. Avoid foaming
if possible.
10) Spin 12,000 rpm/16,000g SW41ti rotor 15 min.
11) Load the supernatant onto a 40% sucrose cushion and spin
at 26,000 rpm for 2.5 hr. 40C in a Beckman SW41ti rotor.
12) Resuspend the pellet in 3 ml of PBS plus 1 ml of 40% CsCl
13) (final concentration 10%) and layer on top of 40% CsCl
14) In SW41ti tube.
15) Spin at 28,000 rpm for 20hr. at 40C.
16) Use an 18ga needle and 5 ml syringe to remove faint band 2/3
of the way down the tube being careful not to disturb the
gradient. (Should be 1-3 ml)
17) Dialysis against 3 X 1 liter changes of PBS that is 1M NaCl
overnight.
18) Spin capsid prep to remove any insoluble material.
The band may be faint, I hold the tube against a black background when pulling the
band.
Article I.
Breaking Buffer:
1M NaCl 0.02M Tris-HCl pH 7.5
SF900 II SFM Media plus 10% FBS plus Gentamicin
Invitrogen cat. 10902-096
Gentamicin
Invitrogen cat. 15710-064
SF9 Cells
Invitrogen cat. 11496-015
Complete protease inhibitor
Roche cat. 1697498