Biomedical Engineering Reference
In-Depth Information
2 At the end of the incubation, add 11.2
μ
lof10
×
proteinase K buffer and 1
μ
lof
proteinase K (20 mg/ml).
3 Incubate the mixture at 42
◦
Cfor1h.
4 In the mixture, add 13
μ
lof3
M
NaAC (pH 7.0), 280
μ
l of ethanol (from
−
20
◦
C) and
0.5
μ
l of blue dextran
q
(20mg/ml).
5 Incubate at
−
20
◦
Catleastfor30min.
6 Centrifuge at
>
12 000 rpm, 4
◦
C for 20min; discard the supernatant.
7 Wash the pellet with 70% ethanol (from
−
20
◦
C); discard the supernatant.
8 Dry the pellet in air.
9 Dissolve the pellet in 10
μ
l of nuclease-free water.
10 Make a serial dilution of the pellet (from purified rAAV) (mostly dilute by 1 : 10 and
1 : 100) for subsequent QC-PCR.
11 Each test sample (purified virus) normally to set four or eight reactions
r
as
follows:
Internal standard control
Purified virus (unknown PP)
(1)
1
μ
l (200 pg/
μ
l)
1
μ
l (1 : 100 dilution)
(2)
1
μ
l (100 pg/
μ
l)
1
μ
l (1 : 100 dilution)
(3)
1
μ
l (20 pg/
μ
l)
1
μ
l (1 : 100 dilution)
(4)
1
μ
l (10 pg/
μ
l)
1
μ
l (1 : 100 dilution)
(5)
1
μ
l (200 pg/
μ
l)
1
μ
l (1 : 10 dilution)
(6)
1
μ
l (100 pg/
μ
l)
1
μ
l (1 : 10 dilution)
(7)
1
μ
l (20 pg/
μ
l)
1
μ
l (1 : 10 dilution)
(8)
1
μ
l (10 pg/
μ
l)
1
μ
l (1 : 10 dilution)
12 Add three controls: one positive control of full-length gene, a second positive control of
ISC and one negative control.
13 PCR reactions mixture: a 25
μ
l reaction volume contains 1
×
PCR buffer, 1.5m
M
MgCl
2
,
0.2
μM
dNTPs, 0.2
μM
of template forward primer, 0.2
μM
template reverse primer and 1
unit of Taq polymerase.
14 Amplification: 4 min at 94
◦
C; 25 cycles of 30 s at 94
◦
C, 30 s at 53
◦
C (annealing),
1minat72
◦
C and a final extension period of 10min at 72
◦
Cinathermal
cycler.
15 Electrophoresis: 10
μ
l of amplification products (with 2
μ
lof6
×
loading buffer) were
analyzed on 1.5% agarose stained with ethidium bromide.
16 The image of the stained gel was taken using an Epi Chemi II darkroom.
17 The densities of the target and competitor bands in each lane were measured using
VisionWorks
®
LS Analysis Software.
