Editing Lentiviral concentration by anion-exchange chromatography (section)

= Protocol 2<br> =

Lentiviral concentration by anion exchange chromatography using Mustang Q anion exchange membrane cartridges

<br>1. Sterilize six Ultra-clear SW28 centrifuge tubes by spraying them down with a 70% ethanol solution. Place tubes into a laminar flow hood with the UV light on for 30 min.

2. Set up two Acrodisk units with Mustang Q membranes. Assemble the two Acrodisk units in a tandem manner.

<br>3. Pipette 25 ml of PBS without Ca/Mg into each of the six sterilized Ultra-clear SW28 centrifuge tubes.

4. Add 20 ml of buffer C to 204 ml of the vector-containing cell culture supernatant after passing it through a 250-ml 0.45-uM PES filter flask.

5. To load the lentivirus-containing sample onto the tandem Acrodisk units, draw up about 32 ml of the sample into a 30-ml syringe and pass the sample through the tandem Acrodisk units. Avoid the formation of bubbles during loading of the sample.

<br>6. To wash the sample adsorbed to the Acrodisk units, attach a 5-ml syringe containing 3 ml of buffer A. Push the buffer through the two Acrodisk units. Avoid the formation of bubbles.

7. To elute the vectors, attach a 10-ml syringe containing 10 ml of buffer B and push the buffer through. Collect the eluate in an Ultra-clear SW28 centrifuge tube containing 25 ml of PBS.

8. Regenerate the Acrodisks by passing 5 ml of a 1 N NaOH solution through followed by 5 ml of buffer A. 

9. Load another 32 ml of the vector-containing supernatant, repeat this cycle four more times until all of the vector-containing supernatant has been processed. The six Ultra-clear SW28 centrifuge tubes will each contain 10 ml of vector-containing eluate and 25 ml of PBS.

10. Centrifuge the samples using ultracentrifuge.

11. Resuspend the pellets.

12. Aliquot resuspended sample in screw-cap microfuge tubes in 25- to 100-ul portions.

<br>13. Snap-freeze tubes in crushed dry ice and store at -80 oC.

Buffer Aâ0.15 M NaCl, 25 mM Tris-HCl, pH 8.0;<br>Buffer Bâ1.5 M NaCl, 25 mM Tris-HCl, pH 8.0; <br>Buffer Câ1.5 M NaCl, 250 mM Tris-HCl, pH 8.0.<br> These buffers can be stored at 4 1C for up to 1 month.

PES filter units, 0.45 mm (Corning, cat. no. 430768)<br>PES filter units, 0.22 mm (Corning, cat. no. 431096)<br>Ultra-clear SW28 centrifuge tubes (Beckman, cat. no. 344058)<br>Acrodisk units with Mustang Q membrane, 0.8 mm, 25 mm (Pall, cat. no. MSTG25Q6)<br>[[Category:Protocols]]<br>