6
7.
Collect the AAV supernatant by centrifugation at 10,000 x g for 10 minutes. Discard the pellet.
8.
Transfer the AAV supernatant to a clean 15 mL conical tube.
9.
Resuspend the AAV Purification Matrix by inverting and shaking. Add 300 µL of matrix to
the centrifuged supernatant.
10.
Mix the supernatant/matrix suspension at
room temperature
for 30 minutes on an orbital
shaker. Do not allow more than 30 minutes and proceed immediately to step 11.
11.
Pellet the Purification Matrix by centrifugation for 10 minutes at 1,000 rpm.
12.
Carefully remove the supernatant and wash the Purification Matrix with 2.5 mL of Purification
Buffer.
13.
Repeat steps 11-12 once more.
14.
Carefully remove the final wash.
15.
Elute the purified AAV from the purification matrix by adding 0.5 mL of Elution Buffer.
16.
Mix at 4ºC for 10 minutes on an orbital shaker.
17.
Collect the elution fraction by centrifugation for 10 minutes at 1,000 rpm.
18.
Carefully remove the elution supernatant.
II. Final Buffer Exchange and Concentration
1.
Assemble a Centrifugal Concentrator unit by inserting the sample reservoir into a recovery
tube.
2.
Apply 0.5 mL of the recovered AAV fraction (step 18 above) to the sample reservoir of the
Centrifugal Concentrator. Cap the concentrator and place into a tabletop microcentrifuge.
Centrifuge for 5 minutes at 2,000 x g. Flow through can be discarded.
3.
Concentrate the AAV fraction until 100 µL remains in the sample reservoir.
4.
Add 400 µL of 1X PBS, or desired final buffer, to the Concentrator and continue to centrifuge
until 100 µL remains. Repeat step 4 once more.
5.
Finally, concentrate until the desired final volume.
6.
Using a clean recovery tube, collect the concentrated AAV sample by inverting the sample
reservoir into the tube and briefly centrifuging to collect all of the liquid.
Содержание ViraBind Series
Страница 3: ...3 Assay Principle...