Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 107

9. Centrifuge it at maximum speed, 4 °C for 5-10 min.
10. Discard the fl ow-through. Add up to 5 ml of sterilized PBS,
then centrifuge it at maximum speed, 4 °C for 5-10 min.
Repeat steps 11 and 12 three times.
11. Bring the volume as you like with PBS. Perform viral DNA
extraction and real-time PCR.
12. Aliquot the virus solution and keep at −80 °C.

1. In hood, transfer 5 µl sample of the virus in 1.5 ml Eppendorf
tube.
2. Prepare DNase I (Promega) mixture.
The volume of reaction mixture is 50 µl/sample. Final concen-
tration of DNase is 50 U/ml.
Samples (collected fractions) are diluted 1:10.

3.2.5 AAV Titration

Viral DNA Extraction

DNase 1,000 U/ml 2.5 µl

Buffer 10× 5.0 µl

H 2 O 37.5 µl

45.0 µl

3. Add 45 µl of DNase I digestion mixture to 5 µl sample and
incubate for 1 h at 37 °C. This digests any viral DNA that has
not been packaged into capsids.
4. Stop digestion by adding 10 µl of 0.1 M EDTA to each
sample.
5. Make proteinase K digestion mixture as follows:

2.5 M NaCl 24 µl (fi nal concentration, 1 M)

10 % (w/v) sarkosyl 6 µl (fi nal concentration, 1 %)

20 mg/ml proteinase K 1 µl (fi nal concentration,

0.2 mg/ml)

Nuclease-free H 2 O 29 µl

60 µl

6. Add 60 µl proteinase K digestion mixture to each sample to
release the viral DNA from the capsid. Incubate for 30 min at
37 °C.
7. Add 120 µl nuclease-free H 2 O for a total of 380 µl.
8. To extract DNA, add 500 µl phenol/chloroform/isoamyl
alcohol and vortex for 1 min.