Transfection to cell using PEI-pro - Lentivirus packaging

1. D-1, seed 293T cells at 4 x 10^6 cells/10mL in a 100mm dish.
2. D+0, 14:00, perform transfection without media change.
3. The final molar ratio of the four vectors should be 1:1:1:1, and total DNA should be 10ug.
4. The condition is satisfied when each DNA is approximately 600fmol.
5. The mass of each DNA at 600fmol differs according to vector length.
6. Therefore, confirm that the final total vector mass is 10ug.
7. Prepare plasmid DNA. All DNA stock concentration is 500ng/uL.
8. Vortex each tube for 5sec.
9. Transfer Tube #1 into Tube #2 and vortex for 5sec. Repeat twice.
10. Incubate at RT for 15min.
11. Transfer the mixture to the cell culture dish.
12. Add dropwise from left to right and top to bottom in zigzag, making small drops.
13. Avoid big drops because they are toxic to cells.
14. D+1, 9:00, change 7.5mL, 75%, of media using serum-free RPMI-1640.
15. D+2, 14:00, harvest all virus-containing supernatant and transfer to storage bottle through 0.45um filter bottle.
16. Concentrate virus by centrifugation using Amicon 100K.
17. Store at -80C.

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