Editing Lentivirus Concentration Ultracentrifuge (section)

== Protocol 2<br>  ==

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

2. Transfer 32-ml aliquots of filtered lentivirus-containing cell culture supernatant into each of the six UV-sterilized Ultra-clear SW28 tubes. 

3. Fill a 10-ml pipette all the way up to the 12-ml mark with a 20% sucrose solution. Insert the pipette all the way to the bottom of the SW28 tube filled with the filtered lentivirus-containing supernatant and gently underlay it by slowly expelling 4 ml of the sucrose solution. Repeat this step for two more of the SW28 tubes using the remaining 8 ml of the 20% sucrose solution from the same 10-ml pipette. For the remaining three tubes, use a new 10-ml pipette and draw up 12 ml of the 20% sucrose solution and proceed in the same way as for the first three tubes. 

4. Adjust the weight of each tube by adding PBS until they are within 0.1 g of each other. 

5. Place all the six tubes into a Beckman SW28 ultracentrifuge rotor. 

6. Centrifuge for 2 h at 25,000 r.p.m. (82,700g) and 4 oC using an ultracentrifuge. 

7. 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. 

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

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

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

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

12. 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. 

13. Aliquot to screw-cap microfuge tubes in 25- to 100-ul portions, snap-frozen in crushed dry ice and stored at -80 oC. 

Alternatively, the pooled viral vector suspension can be further purified by anion exchange chromatography using Acrodisk units with Mustang Q membranes.<br> 

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[[Category:Protocols]]