Biomedical Engineering Reference
In-Depth Information
•
2 l dialysis buffer (10 m
M
Tris-HCl, pH 8.0; 135m
M
NaCl; 1m
M
MgCl
2
)
•
100% glycerol
•
Magnetic stirrer and sterile stir bar
Cryotubes for aliquoting purified recombinant adenovirus.
•
Method
1 Add 3ml low-density CsCl
2
solution (
ρ
=
1.34) to each of two ultracentrifuge
tubes.
e
2 Use a glass Pasteur pipette to underlay with 1.6ml high-density CsCl
2
(
ρ
=
1.4).
3 Carefully add the crude adenovirus lysate on top of the gradient.
4 If necessary, fill the tubes to the top with PBS
++
by underlaying the top of the virus
layer to avoid air bubbles.
5 Ensure tubes are balanced (same mass) and then seal tubes as appropriate.
6 Centrifuge using an ultracentrifuge at 9000
g
in a fixed-angle rotor for 2 h at
18
◦
C.
f
,
g
7 Pierce the top of each tube with a 21-gage needle then using a second sterile 21-gage
needle and syringe carefully remove the white virus layer by piercing the tube
underneath the virus. Avoid taking up surrounding CsCl
2
or perturbing the virus layer as
it is removed.
8 Prepare a second set of CsCl
2
gradient tubes.
9 Dilute purified virus 1 : 2 with PBS and overlay onto new CsCl
2
gradients.
10 Balance and seal tubes as in step 5
11 Centrifuge at 100 000
g
for 18 h at 18
◦
C without brake.
12 Remove discrete band of adenovirus as before in the smallest possible volume.
13 Dialyze against 1 l sterile dialysis buffer for 2 h at room temperature in a laminar safety
cabinet.
14 Dialyze virus for a further 18 h against 1 l fresh dialysis buffer containing 10%
glycerol.
15 Aliquot adenoviral stocks and store at
−
80
◦
C.
16 Carry out titration of stock as in Protocol 11.2.
Notes
e
Prepare ultracentrifuge tubes (two per 15ml of crude lysate) in a safety cabinet by soaking in
70% ethanol for 10min, followed by rinsing with copious amounts of sterilized water. Allow to
dry in the cabinet.
f
Ensure brake is switched off.
g
The virus should form a discrete white layer between the two CsCl2 layers.
