Adeno-Associated Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 18

3. Rinse all wells with 400 ʼl of 0.4 M NaOH/10 mM EDTA
(pH 8.0) solution and apply vacuum to dry the membrane.
4. Disassemble apparatus and rinse membrane in 2× SSC.

1. Prepare hybridization buffer and prewarm to
42 °C. Prehybridization buffer: 1 g NaCl and 1.7 g ECL
blocking agent in 34 ml ECL Gold hybridization buffer.
2. Prewet the membrane in 2 × SSC and place in a roller bottle or
other container. Add appropriate volume of heated hybridiza-
tion buffer (0.0625-0.125 ml/cm 2 ).
3. Prehybridize in the roller for a minimum of 15 min (1 h is
generally used) at 42 °C.
4. Prepare the labeled nucleic acid probe using the kit:
(a) Dilute the DNA to be labeled to a concentration of
10 ng/ʼl in sterile water.
(b) Denature 100 ng of the DNA sample (10 ʼl) by heating
for 5 min in a boiling water bath.
(c) Immediately cool the DNA on ice for 5 min. Spin briefly
in a microcentrifuge.
(d) Add an equal volume (10 ʼl) of DNA labeling reagent to
the cooled DNA and mix gently.
(e) Add 10 ʼl of glutaraldehyde solution, mix and spin in a
microcentrifuge.
(f) Incubate for 10 min at 37 °C (probe may be held on ice
for 10-15 min, or for longer storage of up to 6 months
add 50 % glycerol and store at −20 °C).
5. Remove 1 ml of hybridization solution from the roller bottle
and add labeled probe to it. Then add the probe to the bottle
and hybridize overnight at 42 °C.
6. Prepare the primary wash buffer (25 ml 20× SSC, 360 g Urea,
4 g SDS to 1 l with ddH 2 O. Filter sterilized, stored at room tem-
perature) and preheat to 42 °C. Remove the hybridization solu-
tion from the blot and add the wash buffer (2-5 ml/cm 2 ) and
wash for a maximum of 20 min with gentle agitation at 42 °C.
7. Repeat the wash for 20 min at 42 °C in primary wash buffer.
8. Remove primary wash buffer and wash twice for 5 min each
time at room temperature in 100 ml 2× SSC.
9. Expose Kodak film to the membrane and develop the film.

3.11.4 Membrane
Hybridization Using ECL
Nucleic Acid Direct
Labeling and
Detection System

Compare the intensities of the dots obtained for the virus to one
of the standards (twofold serial dilutions of known quantities of
the AAV vector from approx. 80-0.3125 ng are prepared for
comparison). One may need to estimate in between the standards
(e.g., 10 ʼl viral DNA dot might appear to be between the 5 and
10 ng standard, therefore estimate this to be 8 ng).

3.11.5 Calculation

of Titers