Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 36

a series of tenfold serial dilutions in LRS (20 ng/μl to
0.002 ng/μl).
Prepare two dilutions of the DNase digested viral sample
(already at 1:10 dilution):
2 μl of viral DNA into 18 μl of LRS (1/100 dilution)
1 μl of viral DNA into 49 μl of LRS (1/500 dilution)
Set up a PCR master mixture for the appropriate number
of samples to be amplified. Include a negative control
(water only). Combine 10 μl of SYBR green Jumpstart
Taq Ready mix, 4.8 μl of distilled water, 0.5 μl
forward primer at 20 μM, 0.5 μl reverse primer at
20 μM, and 3.2 μl of MgCl 2 25 mM for a total volume
of 20 μl per reaction.
Place all the capillary tubes in the prechilled (4 °C) lead
box.
Dispense 19 μl of the PCR mixture to each tube and
add 1 μl of diluted viral DNA or 1 μl of each DNA
standard.
Spin the capillary tubes at 1,000 rpm for 10 s.
Perform thermal cycling using optimized cycling condi-
tions. PCR cycling parameters will vary depending
on the melting temperature of the primer set and on
the length of the PCR product.
Analyze the PCR amplification products on a 2 % agarose
gel.
The virus concentration is determined by extrapolation to
the standard curve by using the Light Cycler analysis
software. Calculate the final viral titer in gcp/ml.

23

×

Y
N

× ×
×× ×

6 022 10
110

.

gcptiter = dilution factor × 100 , 0

9

650

where Y is the virus concentration in ng and N is the
length of the DNA template in base pair.
(γ) Dot blot assay
This assay is used to determine the genome-containing
particle titer using transgene-specific DNA probes.
Prepare a standard curve with linearized rAAV plasmid
DNA. Dilute the digested DNA into TE at a concen-
tration of 50 ng/10 μl and make a series of twofold
serial dilutions (50 ng/10 μl to 0.024 ng/10 μl).
Take 25 μl of the DNase digested viral sample and prepare
five serial twofold dilutions in TE.