Biomedical Engineering Reference
In-Depth Information
12 Centrifuge the eluted virus at maximum 2000
g
at 10
◦
C until the volume is reduced to
300-500
μ
l.
13 Add 5ml of 1
×
PBS (or 5ml of Lactated Ringer's solution) into the filter, centrifuge at
2000
g
at 10
◦
C until the volume is around 300-500
μ
l.
14 Repeat step 13 two more times and transfer the concentrated virus (300-500
μ
l) to a
1.5ml tube.
15 Store the concentrated virus at 4
◦
C for characterization of the purified rAAV.
n
Notes
l
Shake heparin-agarose vigorously and add it into the column immediately.
m
Use flat end of a 25 ml sterile pipette to push the filter into the column until touching the
heparin-agarose without any air bubbles.
n
Store the concentrated virus at 4
◦
C for short periods and store at
−
80
◦
C in small aliquots for
the long term.
PROTOCOL 12.5 Titration of AAV: Determining rAAV Physical
Particles by QC-PCR
o
Equipment and reagents
•
10
×
DNase buffer: 500m
M
Tris pH 7.5 and 100m
M
MgCl
2
•
DNase I
•
10
×
proteinase K buffer: 100m
M
Tris pH 8.0, 100m
M
EDTA and 10% SDS
•
Proteinase K (20mg/ml)
•
3
M
NaAC pH 7.0
•
Ethanol (absolute - 200 proof)
•
Blue dextran (Sigma): 20mg/ml
•
Internal standard control (ISC)
p
•
PCR reagents: 10
×
PCR buffer, 50m
M
MgCl
2
,10m
M
dNTPs, 10
μM
template forward
primer, 10
μM
template reverse primer and Taq polymerase
•
Nuclease-free water
•
Water bath
•
Thermal cycler (iCycler, Bio-Bad)
Epi Chemi II darkroom (UVP).
•
Method
1DNaseIdigestion:5
μ
l of purified rAAV virus, 10
μ
lof10
×
DNase buffer with 10 U of
DNAse I in 100
μ
l of reaction mixture, incubate at 37
◦
Cfor1h.
