Lipofectamine 3000 transfection
Default transfection reagent for "hard" cell lines: HeLa, U2OS, primary cells, iPSCs (use Stem-specific reagent there). For HEK293T, prefer cheaper PEI.
You'll need
- Lipofectamine 3000 reagent
- P3000 reagent (the new "enhancer", comes with the kit)
- OptiMEM I reduced-serum medium
- DNA at > 100 ng/µL, endotoxin-free
Volumes per well/dish
| Format | DNA | P3000 | Lipo 3000 | OptiMEM (each tube) |
|---|---|---|---|---|
| 96-well | 100 ng | 0.2 µL | 0.15–0.3 µL | 5 µL |
| 24-well | 500 ng | 1 µL | 0.75–1.5 µL | 25 µL |
| 12-well | 1 µg | 2 µL | 1.5–3 µL | 50 µL |
| 6-well | 2.5 µg | 5 µL | 3.75–7.5 µL | 125 µL |
| 10 cm dish | 10 µg | 20 µL | 15–30 µL | 500 µL |
Procedure (one 6-well format example)
- Day −1: Plate cells to reach ~70–90% confluence at transfection.
- Day 0 — Tube A: Dilute Lipofectamine 3000 in OptiMEM. Mix briefly.
- 125 µL OptiMEM + 5 µL Lipofectamine 3000
- Tube B: Dilute DNA + P3000 in OptiMEM.
- 125 µL OptiMEM + 2.5 µg DNA + 5 µL P3000 (use 2 µL P3000 per µg DNA)
- Combine: add the entire Tube B into Tube A. Pipette gently up and down to mix.
- Incubate at room temperature 10–15 min.
- Add the 250 µL complex dropwise to the well. Rock to distribute.
- Return to incubator. Optional: medium change at 4–6 h to reduce toxicity. Most cell types tolerate the reagent for 24+ h.
- Assay 24–72 h post-transfection.
Tips
- P3000 is critical — without it efficiency drops ~30%.
- For sensitive cell types (primary, iPSCs), titrate Lipo 3000 down — toxicity often limits readout, not transfection efficiency.
- Plasmid quality matters more here than for PEI. Endotoxin-free preparations are recommended.
- Don't include antibiotics in the transfection medium.
- For RNA delivery (mRNA, siRNA), use Lipofectamine MessengerMAX or RNAiMAX instead.