Lipofectamine Plus
| COMPONENT | VOLUME (PER WELL) | X | # WELLS | = | TOTAL VOLUME |
| Tube 1 |
| Plasmid: | 1 µl | X | _____ | = | _____ |
| SFM: | 99 µL | X | _____ | = | _____ |
| TOTAL | 100 ml | X | _____ | = | _____ |
| Plus Reagent | 15 ml | X | _____ | = | _____ |
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| COMPONENT | VOLUME (PER WELL) | X | # WELLS | = | TOTAL VOLUME |
| Tube 2 |
| SFM: | 92 µl | X | _____ | = | _____ |
| Lipofectamine | 8 µL | X | _____ | = | _____ |
| TOTAL | 100ml | X | _____ | = | _____ |
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Proceedure:
1. Day before transfection, plate 150K cells per well of a 6-well plate
2. Grow overnight
3. Next day, dilute DNA (0.5µg/µl) in Serum- and antibiotic-free media in a 15ml tube
4. MIX
5. Add Plus Reagent and MIX
6. Incubate at room temperature for 15 min.
7. Dilute Lipofectamine in Serum- and antibiotic-free media in a different 15ml tube
Mix gently
8. Combine Lipofectamine and DNA
Pipette in and out 5-times
9. Incubate 15 min at room temperature
During:
Aspirate media and wash 1 X DPBS
Add 0.8ml Serum- and antibiotic-free media to each well
10. Add 200ml of the transfection solution dropwise to each plate
11. Incubate plates at 37¡C for 5 hr
12. Add 1ml growth media
13. 17-24 hours later change rinse 1 X with PBS and add 2ml growth media
14. 17-24 hours later switch to differentiation media
15. Collect the cells 4-6 days later