Biomedical Engineering Reference
In-Depth Information
6 Add 2.0 ml of lysis buffer into each 50ml conical tube, up and down four to five times to
re-suspend pellets by 5 ml pipette, combine all of pellets in to one of 50 ml conical
tube
7 Transfer 2 ml more lysis buffer to five of the 50 ml conical tubes (one by one to
resuspend the remaining pellets and pour all pellets suspension (about 15ml) in one of
the 50ml conical tubes.
8 Store cell pellets suspension in
−
20
◦
C freezer for extraction and clarification of
virus.
PROTOCOL 12.3 Purification of AAV: Iodixanol Purification
Equipment and reagents
•
Dry ice
•
Ethanol
•
Benzonase (Sigma)
•
Oak Ridge tubes
•
Quick Seal 50ml ultracentrifuge tubes (Beckman Coulter)
•
12 cm
3
syringes
•
19-GA and 16-GA needles
•
100
μ
l micro-capillary pipettes (Kimble)
•
Parafilm
•
Iodixanol 60% (w/v) (Accurate Chemical & Scientific Corp.)
•
5
M
NaCl
•
1
×
PBS-MK: 1
×
PBS, 1 m
M
MgCl
2
and 2.5 m
M
KCl
•
Phenol red (Sigma)
•
Lysis buffer: 150m
M
NaCl and 50m
M
Tris (pH 8.4)
•
Heat sealer
•
70 Ti/70.1 Ti rotor (Beckman Coulter)
•
Optima L-90K ultracentrifuge (Beckman Coulter).
Method
1 Extract virus from the cells by freezing (dry ice-ethanol) and thawing (at 37
◦
C) at least
three times.
f
2 Add benzonase into about 15ml cell lysate from harvesting (50 units/ml) and incubate
at 37
◦
C for 30min.
3 Transfer the 15ml lysates to one Oak Ringe tube and centrifuge at 400 rpm (2000
g
)and
4
◦
C for 20min.
