Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 37

Add 200 μl of 0.4 M NaOH, 10 mM EDTA to 10 μl of
diluted viral DNA or to 10 μl of each standard.
Perform each sample and control in duplicate.
Set up the slot blot apparatus with two pieces of Whatman
paper and a nylon membrane prewetted in H 2 O.
Incubate viral DNA and standards at 100 °C for 5 min.
Load standards and viral DNA into the wells, apply
vacuum.
Wash each well with 200 μl of 0.4 M NaOH and 10 mM
EDTA.
Disassemble the slot blot apparatus. Rinse the membrane
in 2× SSC. Air-dry the membrane.
Immobilize DNA on membrane using a UV crosslinker
setup at 1,200 × 100 μJ/cm 2 .
Prehybridize the membrane in 0.75× NW buffer for at
least 2 h in a hybridization incubator at 65 °C.
Label a rAAV-specific 32 P-probe using a random primer
labeling kit.
Dilute 25 ng of DNA in H 2 O to a final volume of 42 μl.
Incubate at 100 °C for 5 min; chill on ice immedi-
ately for 1 min. Add 3 μl of α- 32 PdCTP and 3 μl of
Magenta DNA polymerase (4 U/μl). Incubate the
reaction at 37 °C for 20 min and stop the labeling
reaction by adding 2 μl stop mix.
To remove unincorporated nucleotides, centrifuge a
Sephadex G50 spin column at 1,100×g for 2 min.
Load the probe into the column. Centrifuge at
1,100×g for 4 min and collect the flow through.
Count radioactivity and calculate the specific activity
of the probe.
Denature the radiolabeled probe (10.10 6 cpm) for 5 min
at 100 °C. Add immediately to the prehybridization
solution.
Hybridize overnight at 65 °C in a hybridization
incubator.
Pour off the hybridization solution and discard into the
waste container.
Wash the membranes with 0.5×, 0.1× NW buffer for
20 min each at 65 °C.
Air-dry the membrane and wrap with Saran paper.
Expose the membrane to a storage phosphor screen for
few hours at room temperature. Scan the exposed