Lentivirus-Production-293T

1. 24 hours before transfection, split cells into  four 15cm plates/dishes for a confluency of ~80% the next day, add 25 ml of complete DMEM medium (10% FBS, with P/S).

2. Transfection

For each plate:

In a 50 ml sterile tube, add 180 ul GeneExpresso Max into 600 ul serum-free  DMEM medium--->Mix A

In a 50 ml of sterile tube, add 30 ug of lentivirus vector with GOI, then 30 ug V+G packaging vector mix (V:G, 1:2, or 1:1) to 600 ul DMEM medium--->Mix B.

Mix by pipetting using a filtered tip.Transfer Mix A to Mix B, mix by pipetting. Incubate 15-20 minutes at room temperature. Pipette transfer ~1.2 ml mix onto one plate.

4 plates:

720 ul GeneExpresso Max in 2.4 ml serum free DMEM

120 ug of lentivirus vector + 120 ug V+G packaging mix in 2.4 ml serum free DMEM.

3. Optional Replace medium 48 hours after transfection (do not discard the medium, put the conditioned medium into clean 50 ml polypropylene tubes). Store conditioned medium in cold room. Use  medium containing 2% serum and 6 μM sodium butyrate

4. Collect supernatant2 and  4 days after transfection, store virus containing-medium in cold room. Add 25 ml fresh complete medium.

or collect supernatant on day 3

5. Collect supernatant 6 days after transfection. Discard plates.

6. Combine supernatant and precipitate the virus using PEG.

Use psPAX2 (503)+ VSVG (505)

	15cm dish	10cm dish	6 well
psPAX2 (packaging plasmid)	15 ug	6 ug	1 ug
pVSVG (envelope plasmid)	7.5 ug	3 ug	0.5 ug
pLVX or pLenti	22.5 ug	10 ug	1.7 ug
Total DNA	45	19	3.2