Adeno-Associated Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 19

AAV-GFP: 6.258 kb
1 bp = 650 so 650 × 6,258 = 4.0677 × 10 6 (molecular weight of
this plasmid)
To obtain the number of molecules/ng, Avogadro's number is
divided by the molecular weight of this plasmid and 10 9 .

23

60210
4 0677 10

. ´

´´ =

i.e., 1 4799 10 . ´

8

mol ng /

6 9

. 10

Therefore, if the 10 ʼl viral sample contains 8 ng AAV/GFP,
the titer will be:

8

814799 10
10

´ . ´

( )

= 1 1839 10 . ´

8

DNase resistantparticles drpm /

Since AAV particles contain single-stranded DNA, while the stan-
dards are double-stranded DNA, the titer calculated above needs
to be doubled to give the true titer; i.e., 2.3678 × 10 8 drp/ʼl or
2.3678 × 10 11 drp/ml.

1. 1 ʼl sample + 99 ʼl buffer (DNase buffer 1): total 100 ʼl.
2. Add 350 units/sample of DNase1.
3. Incubate in a PCR machine for 30 min at 37 °C, then 10 min
at 95 °C.
4. Spin the samples for a few seconds.
5. Add 1 ʼl Proteinase K (10 mg/ml) to each sample.
6. Incubate in a PCR machine for 60 min at 50 °C, then 20 min
at 95 °C, and finally cool to 4 °C.
7. Store at −20 °C or use straight away.

3.12 Titration of AAV
by Real-Time PCR

3.12.1 Sample
Preparation for
Real-Time PCR

1. The total volume per reaction is 25 ʼl.
The master mix contains the following components:
Taqman mix (Applied Biosystems): 12.5 ʼl
Distilled water: 7.25 ʼl
Forward primer: 1 ʼl (inal concentration: 0.3 ʼM)
Reverse primer: 1 ʼl (inal concentration: 0.3 ʼM)
Probe: 0.75 ʼl (inal concentration: 0.2 ʼM)
Note: All standards and samples should be in triplicates, there-
fore, for a reaction containing 4 samples and 6 standards:
375 ʼl of Taqman mix
30 ʼl of forward primer
30 ʼl of reverse primer
22.5 ʼl of probe
217.5 ʼl of water

3.12.2 Taqman PCR

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