Editing 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&nbsp; of Buffer '''D'''.

3. Load lentivirus suspension onto the <strike>tandem </strike>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.

<br/>'''Buffer A'''

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

'''Buffer B'''<br/>1.5 M NaCl, 25 mM Tris-HCl, pH 8.0;

'''Buffer C'''<br/>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.
[[Category:Protocols]]