Gene Therapy for Epilepsies - Gene Delivery and Therapy for Neurological Disorders - page 251

7. Dilute the viral DNA in 1:50 by adding 2 μl of viral DNA
sample to 98 μl of TE buffer.
8. Make up a PCR master mix. First, calculate the number of
samples, standards, and controls. Perform PCR in triplicate on
each sample and one set of standards in duplicate and include
two water-only (no template) controls and two plasmid con-
trols. Multiply the number of total samples by 1.3. This is to
allow loss of volume from pipetting. Multiply the volume
required for a single sample as given below by this multiplica-
tion factor to obtain the final amount required for the
master mix.
For 1 reaction (total volume of 20 μl):
2× SYBR Green mix, 12.5 μl
Forward primer, 0.5 μl

Reverse primer, 0.5 μl

H 2 O, 6.5 μl
9. Mix well by pipetting up and down five times, and then aliquot
20 μl of the master mix into the bottom of each well of a
96-well optical reaction plate.
10. Thaw 1 set of standard curves (prepared ahead of time, see
Sect. 4 ). Mix well and centrifuge at 200 × g for approximately
5 s.
11. Add 5 μl of each sample, standard, or water per well of the
96-well optical reaction plate already containing the SYBR
Green PCR reaction mix. An example of plate setup with 1
AAV test article, triplicate sample preparation, and triplicate
PCR reaction per sample preparation replicate is given below.

S=AAV test article
C=AAV control sample
SC=standard curve
P=plasmid control

SC 1 × 10 7 SC 1 × 10 7 C1 C1 C1

SC 1 × 10 6 SC 1 × 10 6 C2 C2 C2

SC 1 × 10 5 SC 1 × 10 5 C3 C3 C3

SC 1 × 10 4 SC 1 × 10 4 S1 S1 S1

H 2 O H 2 O S2 S2 S2

P P S3 S3 S3

12. Place an optical adhesive cover onto the plate and ensure it is
fully sealed using the roller.
13. Shake well to mix the sample.