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Virus Purification - Adenovirus mini kit

Harvest supernatant from adenovirus-infected cells
  1. Centrifuge the adenovirus-infected culture media at 3,000 rpm for 10 minutes. Filter the supernatant through a 0.45 μm filter unit to remove cells and debris.
  • Supernatant from one to two T75 flasks can be processed per column. Up to 1 x1012 virus particles can be purified per column.
  1. The supernatant is ready for purification. The supernatant can also be stored at -80oC for future purification.  
Equilibrate the column
  • Dilute the 10 x Wash Buffer with ddH2O to 1 x Wash Buffer.
  • Dilute the 2 x Elution Buffer with ddH2O to 1 x Elution Buffer.
  1. Set the column in a 15 mLcentrifuge tube and spin in a swing bucket rotor at 300 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 ddH2O and then 5 mL 1x Wash Buffer.   
  • Centrifugation can help remove the bubbles created during shipping.
  • A swing-bucket rotor is preferred for centrifugation.
  • If the flow-through is too slow, the other alternative is to set the column in a 15 mL conical tube and centrifuge at 400 x g for 5 minutes.
  • 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.
Load the column
  1. Load 5 mL of supernatant to the column and let the supernatant gradually run through the column. Transfer the flow through to another clean tube. Keep loading till all samples pass through the column. Reload the flow through to ensure maximal viral particle binding. 
  • If the flow rate gets noticeably slow, cap (the press-on cap to the bottom and the screw cap to the top) and invert the column to mix the supernatant and resin well. Rock the sample for 5 minutes in a shaker platform. Take off the press-on cap and put the column into 15 mL tube. Centrifuge at 300 x g for 2 minutes. Transfer the flow through to another clean tube if reloading is needed. Keep loading the supernatant till all samples pass through the column.      
Wash the column and elute the adenovirus
  1. Wash the column with 5 mL 1 x Wash Buffer. Repeat once. This step can be performed either by gravity flow or centrifugation at 400 x g for 5 minutes.

  2. Elute the virus by applying 2-4 mL Elution Buffer. Collect the elution in tubes at 1 mL each. Measure the OD260 and OD280 of each fraction to identify the virus pool.

 Desalting
  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. 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 Concentrate volume
    10 min 176 μL
    15 min 76 μL
    20 min 58 μL
  • Typical Concentration Volume Vs. Spin Time (35o Fixed angle rotor RT, 4 mL starting volume) for 100K centrifugal filter device
    Spin time Concentrate volume
    10 min 97 μL
    15 min 54 μL
    20 min 35 μL 
 
  1. Sterilize the purified virus by passing through a 0.22 μm syringe filter.The filter unit retains some virus particles after filtration.Elute the filter unit with 300 µL of desired low salt buffer to collect the retained virus particles.

  2.  Aliquot and store the final purified virus at -80°C.

  3.  

Regeneration of the column 

Upon completion of the purification, add 5 mL Regeneration Buffer to the column by gravity flow and then add 5 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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