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Virus Purification - Retrovirus purification mini kit

Harvesting supernatant from retrovirus-infected cells (1-2 T75 flask or equivalent per column)

  1. Centrifuge the Retrovirus-infected culture media at 3,000 rpm for 10 minutes. Filter the supernatant through a 0.45 μm filter unit. Supernatant from one to two T75 flasks can be processed per column. Up to 3 x1010 virus particles can be purified per column. The supernatant can also be stored at -80°C for future purification.

Equilibrate the column

  • Dilute the 10x Wash Buffer with ddH2O to 1x Wash Buffer.
  • Dilute the 2x Elution Buffer with ddH2O to 1x Elution Buffer.
  1. Set the column in a 15 mL centrifuge tube and spin at 600 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 600 x g for 1 minute.
  • There’s a press-on cap supplied in the kit for the bottom of the column to stop the flow.
  • If the flow-through is too slow, make sure to remove any visible bubbles (See trouble shooting on page 6).

Load retrovirus-containing supernatant to the column

  1. Load the supernatant to the column and let the supernatant gradually run through the column. Collect the flow through and reload to the same column one more time to ensure maximal viral particle binding.

  • If the gravity flow through rate gets noticeably slow during loading or reloading of the supernatant, set the column in a 15 mL conical tube and centrifuge at 300 x g for 1 minute. Repeat two times to ensure maximal viral particle binding.
  • The visible and invisible bubbles in the resin bed normally cause the slow flow rate.

Wash the column and elute the Retrovirus

  1. Wash the column with 5 mL Wash Buffer. Repeat once. This step can be performed either by gravity flow or centrifugation at 600 x g.

  2. Elute the retrovirusby 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.This step can also be processed by centrifugation at 300 x g with 1 mL Elution Buffer, repeat to collect other elutions.

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 (35Fixed 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.

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 Wash Buffer. Press on the cap to the bottom. Wrap the column with parafilm in a zip block bag and store at 4℃.

 

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