Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 34

(b) Benzonase treatment
To each 10 ml of crude lysate, add 2 μl of 4.9 M MgCl 2 , vortex.
Add 1,500 U of benzonase, vortex.
Incubate at 37 °C for 30 min. Mix by swirling every 5 min.
Clarify the crude lysate by pelleting the cell debris in a tabletop
centrifuge at 2,500 × g for 20 min at 20 °C. Transfer the
lysate (supernatant) into a new 50-ml tube.
(c) Iodixanol density gradient
Pour four-step gradients into open-toped Optiseal polyallomer
tubes starting with the least dense solution:
- 7.3 ml of 15 % iodixanol/1 M NaCl.
- 4.9 ml of 25 % iodixanol.
- 4 ml of 40 % iodixanol.
- 4 ml of 60 % iodixanol.
Carefully overlay the clarified crude lysate onto the iodixanol gra-
dient. Fill the centrifuge tube to the neck with crude lysate.
Cap and balance all tubes using an analytical scale. Carefully
place the tubes into the 70Ti rotor and cover each tube with
a tube spacer.
Ultra-centrifuge the samples at 69,000 rpm for 1 h at 18 °C. Set
for fast acceleration and no brake.
Once the gradient run is completed, carefully extract purified
virus from each tube. Place a clamp stand inside the bio-
safety cabinet. Hold the tube in the clamp, remove the cap,
and clean the outside of the tube with a sterile Alcohol wipe.
Insert an 18G × 1½ in. needle underneath the 60-40 % interface
and aspirate half of the 40 % layer.
Collect the virus into a new 15-ml tube.
(d) Virus concentration
Rinse a 100 K cut off concentrator with 10 ml of Lactated
Ringer's solution (LRS) to remove traces of glycerin and
sodium azide. Spin for 5 min at 2,000 rpm at room tem-
perature. Empty the filtrate container. Aspirate any remain-
ing LRS in the concentrator.
Dilute the virus with at least 3 volumes of LRS, load virus into the
concentrator. Rinse the virus tube with 1 ml of LRS and add
to the concentrator. Fill up to 20 ml with LRS. Centrifuge at
2,000 rpm and bring the volume down to 1 ml.
Add 20 ml of LRS to wash virus. Centrifuge at 2,000 rpm and
bring the volume down to 1 ml.
Repeat wash step two more times and bring down the final
volume to 500 μl.