Biology Reference
In-Depth Information
3.4 HC-Ad Vector
Purifi cation
Purifi cation of HC-Ad vectors is performed by double CsCl
banding and subsequent desalting. The fi rst CsCl banding is based
on a discontinuous CsCl gradient, removes >90 % of cellular
impurities, and concentrates the vector particles. The second CsCl
banding is based on a continuous CsCl gradient. To obtain HC-Ad
vector preparations with high purity it is mandatory to have at least
one discontinuous CsCl step gradient. Extra high purity can be
obtained by performing two subsequent discontinuous CsCl
gradients followed by one continuous gradient. The steps given in
the following protocol refer to purifi cation of HC-Ad vector
particles from twelve 150-mm dishes (approximately 2-3E8 cells).
For upscaling
see
Note 14
.
1. Prepare CsCl solutions with densities of 1.27 g/mL, 1.34 g/
mL, and 1.42 g/mL, respectively, in HEPES 50 mM pH 8.0
(
see
Note 15
).
2. Release the vector particles from the cells obtained in
step 32
of the amplifi cation protocol by three repeated freeze-thaw
cycles in liquid nitrogen and a water bath (37 °C).
3. Spin down cell debris by centrifugation at 5,000 ×
g
for 10 min
at 4 °C and transfer the supernatant containing the released
vector particles into a new tube. Centrifuge again at 5,000 ×
g
for 10 min at 4 °C (
see
Note 16
).
4. Pipette 3 mL of the sterile-fi ltered CsCl solution with a density
of 1.42 g/mL into a UltraClear centrifuge tube (14 × 89 mm,
12.8 mL total volume).
5. Carefully overlay with 5 mL of a CsCl solution with a density
of 1.27 g/mL.
6. Carefully overlay with the cleared supernatant obtained in
step
3
of the purifi cation protocol.
7. Carefully fi ll up the remainder of the tubes with PBS.
8. Centrifuge in a Beckman SW41Ti rotor at 32,000 rpm
(176,000 ×
g
) for 2 h at 4 °C.
9. After centrifugation a vector band should be visible at the
interface of the 1.42 g/mL and 1.27 g/mL CsCl solutions. In
addition bands comprised of incomplete particles are visible
below the interface of the cell lysate and the 1.27 g/mL CsCl
solution. Pierce the tube with a 2 mL syringe and 18-G needle
below the vector band and slowly retrieve the vector. Take care
to not retrieve more than 2 mL.
10. Transfer the vector into a new UltraClear centrifuge tube
(14 × 89 mm, 12.8 mL total volume).
11. Fill up with the 1.34 g/mL CsCl solution and mix carefully by
pipetting up and down.
