Lentivirus Purification PEG + Q Membrane for in vivo

PEG/NaCl (5x) stock solution

PEG-8000 20%, NaCl 2.5 M. Dissolve 100 g PEG-8000 (20% w/v) and 75 g NaCl (2.5 M) in 400 ml ddH20 and bring to a final volume of 500 ml by stirring at RT. The solution can be autoclaved (optional) but mixing during the cooling period is required to prevent a phase separation. Store at RT.

Final Conc: 4% PEG-8000. 0.5 M NaCl.

Purification of lentivirus by anion exchange chromatography using Mustang Q or Sartius LentiSELECT anion exchange membrane cartridges

1. Set up two Acrodisk Mustang Q membrane disks or one LentiSELECT disk.

2. Precipitate lentivirus from 15 cm plate using PEG. Suspend lentivirus in 20 ml  of Buffer D.

3. Load lentivirus suspension onto the tandem Acrodisk units/LentiSELECT disk using a syringe. Avoid the formation of bubbles during loading of the sample.

4. Wash Acrodisk units with 3 ml of Buffer A using a syringe. Avoid the formation of bubbles.

5. Elute lentivirus with 10 ml of Buffer B using a syringe. Collect the eluate in a 50 ml tube containing 25 ml of PBS.

6. Regenerate Acrodisks by passing 5 ml of a 1 N NaOH solution through followed by 5 ml of Buffer A.

7. Concentrate lentivirus to ~ 2 ml using 100 KDa spin concentrator (3000 rpm for 30 to 60 min). Add 20 ml PBS and concentrate again.

8. Aliquot lentivirus into screw-cap microfuge tubes in in 25 to 100-ul portions.

9. Print labels for virus. Snap-freeze tubes in crushed dry ice and store the tubes at -80 oC.

Buffer A

0.15 M NaCl, 25 mM Tris-HCl, pH 8.0;

Buffer B
1.5 M NaCl, 25 mM Tris-HCl, pH 8.0;

Buffer C
1.5 M NaCl, 250 mM Tris-HCl, pH 8.0.

Buffer D

0.1 M NaCl, 25 mM Tris-HCl, pH 8.0.

Note: If Vivapure LentiSELECT buffers are available, follow Sartius's protocol.