Lentiviral vector packaging and transfection

　　1. Take 293-T cells in good condition, spread them in a six-well plate at 8 x10^5 cells/2.5ml/well, and culture them in a constant temperature incubator at 37°C overnight.

　　2. When the confluence of the cells reaches 60-70%, change the medium and remove the double-antibody P/S in the complete culture medium.

　　3 After changing the medium for 1 hour, transfect with lipo2000 liposomes.

　　4 Prepare the transfection reagent as follows:

　　a) Take two 5 mL sterile centrifuge tubes and label them X-a, X-b,

　　b) Add 250 uL (250 uL) Opti-MEM to each centrifuge tube;

　　c) Add DNA to X-a according to the following ratio: (2 wells)

　　d) Add 20 uL Lipofectamine 2000 to X-b, mix gently, and place at room temperature for 5 minutes;

　　e) Then transfer the corresponding DNA solution in tube a (drop by drop) to tube b, shake gently to mix; leave it at room temperature for 20 minutes

　　f) Add 250 uL of the mixed solution to each well of the 6-well plate, shake it slightly, and return it to the incubator to continue culturing; after 6 hours, replace it with complete culture medium and continue culturing for 48 hours; (the well plate is placed on its side with the suction Clear and add slowly along the wall to prevent 293T from being blown up.)

　　g) Collect the culture supernatant after 48 hours, and add 2.5 mL of complete culture medium to continue culturing;

　　h) Centrifuge 500g of the collected supernatant for 10 minutes;

　　i) Mix the supernatant with 5X lentivirus concentration reagent in a ratio of 4:1, mix gently every 30 minutes; after mixing 3 times, store in a 4-degree refrigerator; generally it can be kept for about a week;

　　j) At 72 hr, continue to collect the culture supernatant, and concentrate and collect the virus according to the above steps; mix the 48 hr supernatant with the 72 hr supernatant and centrifuge (the virus titer will decrease after mixing and can be centrifuged separately) ;Centrifugation conditions are 4 degrees, 4000 g, 25 min;

　　After centrifugation, carefully remove the supernatant. Generally, a white precipitate is visible (sometimes the precipitate is not visible). Add an appropriate volume of PBS (1/10-1/100 of the original supernatant volume); carefully pipette and resuspend; aliquot according to 50 uL/tube. , stored in -80℃ refrigerator.