Editing Lentiviral concentration by anion-exchange chromatography (section)

= Protocol 1<br> =

Lentiviral concentration by anion-exchange chromatography and ultracentrifugation

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

2. Add 27 ml of PBS without Ca/Mg to lentivirus sample.

3. Add 3 ml of buffer C.

4. Pass the sample through the tandem Acrodisk units. Avoid the formation of bubbles.

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

6. 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.

7. Centrifuge for 2 h at 25,000 rpm. (82,700g) and 4 oC using an ultracentrifuge.

8. Carefully remove tubes from the rotor, pour off supernatant and leave tubes on a paper towel in an inverted position for 10 min to allow the residual liquid to drip away from the pellet. Aspirate the remaining medium droplets. There should be a miniscule pellet at the bottom of the tube.

9. Add 100 ml of PBS without Ca/Mg to each pellet.

10. Place the SW28 ultracentrifuge tubes into a 50-ml conical tube and close tube with the lid.

11. Incubate the tubes at 4 oC for 2 h. Vortex very gently every 20 min.

12. Spin the tubes at 500g for 1 min at 25 oC to collect the vector-containing liquid.

13. Using a 200-ul pipette, resuspend the pellet by gently pipetting the liquid up and down. Avoid the formation of bubbles. Combine liquid from all resuspended pellets in a single SW28 ultracentrifuge tube.

14. Transfer resuspended vector sample into screw-cap microfuge tubes in 25- to 100-ul portions.

15. Snap-freeze the 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><br>