Lipofectamine Lentivirus Production (L3000, T25) Introduction Lipofectamine® 3000 is a proprietary formulation for the transfection of nucleic acids (DNA and RNA) into eukaryotic cells.
Materials › ›
Procedure Plate cells for transfection 1. Plate 2 million cells in 5 mL media for each T25 ~24h before transfection.
Transfection 2. HEK293FT cells should be at 90-99% confluency at transfection. 3. Make DNA Mastermix (MM1)
MM1
Number of
1
15
Samples OMEM
625 uL
9375
PMD2.G
1.7 ug
25.5
psPAX
2.6 ug
39
15 uL
225
3.4 ug
51
P3000 Enhancer Target Total (-Target)
644.3
4. Make Lipofectamine Mastermix (MM2)
9664.5
MM2
Number of
1
15
Samples OMEM
625 uL
9375
Lipo3000
17.5 uL
262.5
642.5
9637.5
5. Invert MM2 to mix and add total volume to MM1. 6. Pipette up and down 10x to mix. 7. Incubate for 10 minutes at room temperature. 8. Remove 50% (2.5mL) of the media from the T25 flask. 9. Add 1250uL of DNA-lipid complex (MM1 + MM2) to cells. 10. Incubate for 5 hours at 37C. 11. Replace transfection media with 5 mL of media.
Post transfection 12. Optional: 17-24h after transfection, add caffeine to a final concentration of 2mM. 13. Harvest lentivirus 48 hours after transfection.
Lipofectamine Lentivirus Production (L3000, T25) -
Lipofectamine Lentivirus Production (L3000, T25). Introduction. Lipofectamine® 3000 is a proprietary formulation for the transfection of nucleic acids (DNA and ...
8. Remove 50% (2.5mL) of the media from the T25 flask. 9. Add 1250uL of DNA-lipid complex (MM1 + MM2) to cells. 10. Incubate for 5 hours at 37C. 11. Replace transfection media with 5 mL of media. Post transfection. 12. Optional: 17-24h after transfec
supplemented with 1% BSA to concentrate virus 100x or more. 6. Place ultracentrifuge tube within a tightly sealed 50ml Falcon tube and allow resuspension overnight at 4C for the pellet to completely dissolve. 7. Next morning: Aliquot virus and store
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