www.bioland-sci.com

Virus purification - Lentivirus purification miniprep

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

  • Supernatant from one to two T75 flasks can be processed per column. Up to 3 x1012 virus particles can be purified per column.
  • The supernatant can also be stored at -80°Cfor future purification.
  1. Centrifuge the lentivirus-infected culture media at 3,000 rpm for 10 minutes. Filter the supernatant through a0.45 μmfilter unit.
  2. The supernatant is ready for purification.

Equilibrate the column

Dilute the10x Wash Buffer with ddH2O to1x Wash Buffer.

Dilute the2x Elution Buffer with ddH2O to1x Elution Buffer.

  1. Set the column in a 15 mLcentrifuge 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 with2 mL of 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 the lentivirus-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.

  2. 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.

  3. The visible and invisible bubbles in the resin bed normally cause the slow flow rate.

Wash the column and elute the lentivirus

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

  2. Elute the virus by applying 3-5 mLElution Buffer.Collect the elution in tubes at 1 mLeach. Measure the OD260 and OD280 of each fraction to identify the virus pool.

Desalting

The desalting columncan also be used for buffer exchange instead of dialysis. Sterilize the purified virus by passing through 0.22μmsyringe 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

Regeneration of the column

Upon completion of the purification, add 5 mLRegeneration Bufferto the column by gravity flow and then add5 mL1 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.

Copyright © 2021 Bioland Scientific, LLC. All rights reserved. Powered by Zen Cart. Hosted by Siteground.
FedEx service marks used by permission.