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This experiment introduced the operation steps of concentrating and purifying lentivirus by ultracentrifugation precipitation method.
1. 70% ethanol
2. 20% sucrose solution
3. PBS buffer
1. Ultra-clear SW28 centrifugal tube
2. Ultra clean workbench
3. Ultraviolet lamp
4. 10 ml pipette
5. Beckman SW28 overspeed centrifugal swivel
6. High speed centrifuge
7. 50 ml cone bottom centrifuge tube
8. Low temperature refrigerator
1. Take six Ultra-clear SW28 centrifuge tubes, sterilize them with 70% ethanol, and then put them in the ultra clean workbench and turn on the UV lamp to continue disinfection for 30 minutes.
2. Add about 32 ml of pretreated virus supernatant into each Ultra-clear SW28 centrifuge tube.
3. Take a 10 ml pipette and suck 12 ml of 20% sucrose solution. Insert the pipette all the way to the bottom of the centrifuge tube and slowly inject 4 ml of the sucrose solution. Similarly, the remaining 8 ml of sucrose solution were added to the other two centrifuge tubes. Take another clean pipette and perform the same treatment on the remaining three pipettes.
4. Use PBS to adjust the weight of each tube so that the weight difference between corresponding centrifuge tubes does not exceed 0.1 g.
5. Put all 6 centrifuge tubes into Beckman SW28 overspeed centrifugal swivel in order.
6. Centrifuge for 2 hours at 4℃, 25000 rpm.
7. Carefully remove the centrifuge tubes from the swivel. Pour out the supernatant, and place the centrifuge tube upside down on the paper towel for 10 minutes to drain the remaining supernatant. Suck and discard the remaining droplets. There should be visible sediment at the bottom of the tubes.
8. Add 100 ml PBS free of calcium and magnesium into each tube to wash the sediment.
9. Insert SW28 ultracentrifuge tube into the 50 ml cone bottom centrifuge tube and cover it.
10. Dissolve at 4 ℃ for 2 hours, and shake gently every 20 minutes.
11. Centrifuge at 4 ℃, 500g for 1 minute to concentrate the solution at the bottom of the tube.
12. Use a 200 μl pipette to gently blow to suspend the sediment (avoid foam). Concentrate the liquid in all tubes into a SW28 centrifuge tube.
13. The concentrated virus suspension is divided into 50 μl each and stored in the finished product tube. Store at -80 ℃ after quick freezing with dry ice.
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