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Introduction The ViraTrapTM AAV purification kit is designed for fast and efficient purification all serotypres of rAAV from AAV infected cell culture. Viral particles can be purified from cell culture of 1 to 2 T75 flasks per column. The viruses are first applied to a purification column and then further purified and concentrated through a centrifugal filter. Before Starting Familiar with each step by reading this menu and prepare all materials for the procedure. Follow safety regulations for AAV

Kit Components

Stability The Guaranteed shelf life is 12 months from the date of purchase. Store AAV column at 4°C

Safety considerations

The AAV infected cell culture and the purified virus can be bio-hazardous material and can be infectious to human and animals. All protocols MUST be performed under Bio-Safety working conditions for AAV according to regulations.

Materials required but not supplied ?

Standard TC centrifuge ?

Swing bucket rotor. ?

0.45 μm filter unit ?

Rack holder for column

Harvest AAV infected cells (cells from 1-2 T75 flasks or equivalent) 1. For adherent transfected cells, use a pasteur pipette to remove the culture medium and harvest cells with 3-5 mL PBS. Pellet the cells at 1000 rpm for 10 minutes. Discard supernatant. Resuspend the cells in 3 mL of Buffer B. Make sure there’s no cell clumps remaining after resuspension. 2. Add 30 μL of 100x Nuclease reaction buffer and 5 μL of Nuclease. Mix well by pipetting and incubate at 37°C for 30 minutes. Centrifuge at 600 x g for 15 minutes, transfer the supernatant to a clean tube, further clarify the supernatant through a 0.45 μM filter unit. Add 1 volume of Buffer P to 3 volume of virus lysate (For example, add 1 mL of Buffer P to 3 mL of virus lysate). Mix well and incubate at 4°C overnight. The virus is stable in Buffer P. 3. Centrifuge the sample at 3,000 rpm for 30 minutes (Proceed to step 4 during centrifugation) at 4°C. Carefully aspirate the supernatant. Spin briefly and remove the residual supernatant. The virus containing pellet should be visible. The pellet may appear hazy. Keep the virus on ice and proceed to step “5”. Purification column preparation 4. Inverting the AAV column to resuspend the resin inside the column. Put the column into a 15 mL conical tube and centrifuge at 1000 rpm for 2 minutes. Tear off the breakoff tip on the bottom of the column and place the column into the 15 mL tube. Loosen the cap to allow buffer drain out from the column by gravity. Once the liquid stops dripping, add 4 mL of Buffer S evenly to the column and let it drain out by gravity without drying the column out. Note: A press on cap for the bottom tip of the column is provided for stopping the gravity flow at any time. 5. Dissolve the pellet from step 3 with 4 mL of Buffer S by pipetting and vortexing. Spin the sample at 3000 rpm for 10 min at 4°C and transfer the clear supernatant to a clean tube. 6. Load the sample from step 5 to the reservoir of a centrifugal filter and centrifuge at 3000 rpm for 15-20 minutes till around 300 μL of sample remains in the reservoir. Transfer the sample to a clean vial. Wash the reservoir by 100 μL of Buffer S and transfer the sample to the clean vial. Load the sample to the purification column 7. Apply the sample from step 6 evenly to the AAV column and let it flow into the resin by gravity. Once the sample gets into the resin, proceed to next step. Note: Slowly add the sample dropwise to the resin. Once the entire sample gets into the resin, proceed to next step. Do not let the column dry out. Elute AAV from the purification column 8. Add 4 mL of Buffer ES evenly to the column and collect 4 mL of the flow-through. The virus is in the flow through liquid. Concentration 9. Apply 4 mL of the sample to the reservoir of a centrifugal filter and centrifuge at 3,000 rpm for 10-30 minutes till 500 μL remains in the reservoir. Pipet the solution up and down several times in the reservoir and transfer the virus containing solution to a clean vial. Note: A swing bucket rotor is preferred. Fixed angle rotor requires higher speed of 7000 rpm for 15-20 minutes. Note: Time for centrifugation may vary for different type rotors. Always centrifuge less time and check the liquid level, repeat centrifuge to get to the expected volume. 10. The purified virus is ready for downstream applications. Aliquot and store the purified virus at -80°C. ? Typical concentration volume Vs. spin time (Swing bucket rotor, 3,000 rpm at RT, 4 mL starting volume) for 100K centrifugal filter device Spin time-15 min: concentrate volume 176 μL Spin time-20 min: concentrate volume 76 μL Spin time-25 min: concentrate volume 58 μL ? Typical concentration volume Vs. spin time (35o Fixed angle rotor , 7000 rpm at RT, 4 mL starting volume) for 100K centrifugal filter device Spin time-10 min: concentrate volume 97 μL Spin time-15 min: concentrate volume 54 μL Spin time-20 min: concentrate volume 35 μL Regeneration of the column Upon completion of the purification, add 5 mL of Regeneration Buffer to the column and let the buffer passes through the column by gravity flow. Wash the column by 10 mL of PBS, let the PBS pass through the column by gravity flow. Once the liquid stops dripping, fill the column with 2 ml of PBS. Press on the cap to the bottom. Screw on the cap and wrap the column with parafilm in a zip block bag and store at 4°C.