Recombination virus amplification and purification

293 cells in a 75cm2 square bottle until the density reaches 90%, and the infected cells are added to the appropriate amount of the virus. After 3- 4d, the cells become almost round, and half of the cells float, all cells are collected. About 500g speed centrifugal, discarded.
2. Re-suspend precipitation with sterilized PBS, repeatedly frozen and melted 4 times. 7000g centrifugation 5min. Virus purification requires at least 30 bottles of 75cm2 square bottle cells.
3. CsCl continuous gradient centrifugal purification: 50 ml
centrifugal tube
weighing 4.4g CsCl, adding 8 ml virus lysate on the liquid, mixed, volume of about 10 ml. Transfer to a 12 ml overspeed centrifuge tube (for SW41 turntables), covering approximately 2 ml of mineral oil. After balancing, centrifuge 18-24hr at 32000 rpm at 10 degrees C, using a syringe to suck out the centrifugal virus belt.
(can also CsCl non-continuous gradient centrifugation: 20ml overspeed centrifuge tube slowly add 8ml CSCl 1.4 (53g x 87 mL 10 mM Tris-HCl, pH x 7.4) 9), carefully add 6 mL CsCl 1.2 (26.8 g s 92 mL; 10 mM Tris-HCl, pH s 7.9) above, and carefully add the virus to clear the liquid until the volume reaches 20ml. After balancing, centrifuge 90min (SW28 turntable) at 23000rpm at 4 degrees C, suction of the lower layer of blue-white virus belt with a syringe
4. Virus dialysis desalination: configuration dialysis solution (10 mM TrispH 8.0, 2 mM Mg Cl2, 5% sucrose), sterilization treatment. Dialysis at 4 degrees C, replacement of 3 dialysis solution, can basically remove CsCl, the virus is stored at - 80 degrees C.