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Virus purification - Adenovirus-associated virus mini kit

 I.      Prepare AAV-infected cell lysate (For up to 2 x T75 flasks per column)

1.     For adherent transfected cells, use a pasteur pipette to remove the culture medium and harvest cells with 3-5 mL PBS using a cell scraper.
2.     Pellet the cells at 350 x g for 10 minutes. Cell pellet can be stored at
      -80°C or proceed immediately to the following step.
3.     Resuspend the cell pellet in 3 mL Binding Buffer. Make sure there’s no cell clumps remain after resuspension. This is critical for the release of viral particles.
4.     Add 30 μL of 100x Nuclease Reaction Bufferand 5 μL of Nuclease and incubate the mixture at 37°C for 60 minutes with gentle rocking.
5.     Collect the supernatant with rAAV from the crude by centrifugation at 600 x g for 10 minutes. Further clarify the supernatant by passing through a 0.45 μm sterile syringe filter.
 
II.    Equilibrate the column
6.     Set the column in a 15 mL centrifuge tube and spin at 500 x g for 2 minutes. Hold the column with a clamp or other holders. Twist off the bottom and let the liquid drop by gravity flow. Equilibrate the column with 2 mL of ddH2O and then 5 mLBinding Buffer
·        Centrifugation removes the bubbles created during shipping.
·        A swing-bucket rotor is preferred for centrifugation.
·       If the flow-through gets too slow, the other alternative is to set the column in a 50 mL conical tube and centrifuge at 500 x g for 1 minute.
·        There’s a press-on cap supplied in the kit for the column tip to stop the flow.
·        If the flow-through is too slow, make sure to remove any visible bubbles (See trouble shooting on page 7).
 
III.   Load the AAV-containing supernatant to the column
7.     Load the supernatant to the column and let the lysate gradually run through the column. Collect the flow through and reload to the same column one more time to ensure maximal viral particle binding.
NOTE: If the gravity flow through rate gets noticeably slow during loading or reloading of the lysate, set the column in a 15 mL conical tube and centrifuge at 300 x g for 2 minutes. Repeat two times to ensure maximal viral particle binding.
NOTE:The visible and invisible bubbles in the resin bed created during shipping or from the buffers and lysate during loading normally cause the slow flow rate.
 
IV. Wash off the nonspecific bindings and elute the AAV
8.     Wash the column with 5 mL Binding Buffer. Repeat once. This step can be performed either by gravity flow or centrifugation at 500 x g.
9.     Elute the AAV by applying 3-5 mL Elution Buffer. Collect the elution 1 mL per tube. Measure the OD260 and OD280 of each fraction to identify the virus pool.
Note: This step can be processed by centrifugation at 300 x g with 1 mL Elution Buffer, repeat to collect other elutions.
 
V.    Desalting
The desalting column can also be used for buffer exchange instead of dialysis. Sterilize the purified virus by passing through 0.22 μm syringe filter. Aliquot and store the final purified virus at -80°C.
  1. Place the filter column in to a 15 ml conical tube. Add up to 4 mL of sample (3.5 mL if using a 23 degree fixed angle rotor) onto the filter column. Cap the conical tube.
  2. Place the conical tube into the centrifuge rotor (swinging bucket preferred), counterbalance with a same weight tube.
  3. Spin at 4000 × g for approximately 10–20 min in a swinging bucket rotor. When using a 35 degree fixed angle rotor, spin at maximum 7500 × g for 20 min.
  4. Add PBS or other desired buffer up to the 4 mL mark on the column and spin at 4000 × g for desired time (see below). The viruses are now in the residual buffer at the bottom of the column
  5. To recover the viruses, insert a pipetter into the bottom of the column and withdraw the sample using a side-to-side sweeping motion to ensure total recovery. The ultrafiltrate can be stored in the centrifuge tube with a cap. NOTE: For optimal recovery, remove concentrated sample immediately after centrifugation.
  • Typical residual volume Vs. spin time (Swing bucket rotor, 4,000 g at RT, 4 mL starting volume) for 100K centrifugal filter device
Spin time-10 min: concentrate volume 176 μL
Spin time-15 min: concentrate volume 76 μL
Spin time-20 min: concentrate volume 58 μL
  • Typical Concentration Volume Vs. Spin Time (35o Fixed angle rotor 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
VI.    Regeneration of the column 
Upon completion of the purification, add 5 mL Regeneration Bufferto the column by gravity flow and then add5 mL 1 x Wash Buffer. Press on the cap to the bottom. Wrap the column with parafilm in a zip block bag and store at 4°C. 
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