Before you start
- Seed cells the day before so they are 70–90% confluent at transfection.
- Include a reporter plasmid (e.g. GFP) and an untransfected control.
Reagents and materials
Totals are for 6 wells (+10% extra). Per-well (24-well) amounts are in the middle column.
| Reagent | Per well (24-well) | Total |
|---|---|---|
| Plasmid DNA (endotoxin-low) | 0.5 µg | 0.5 µg |
| Serum-free medium (e.g. Opti-MEM) for DNA | 25 µL | 25 µL |
| Lipid reagent (about 1–3 µL per µg DNA) | 1 µL | 1 µL |
| Serum-free medium for lipid | 25 µL | 25 µL |
| Complete medium in the well | 0.5 mL | 0.5 mL |
Equipment
- Biosafety cabinet
- 24-well plate
- Incubator
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Dilute DNA
Dilute plasmid in serum-free medium.
Dilute lipid
Dilute lipid reagent in serum-free medium (some reagents need 5 min).
Form complexes
Combine diluted DNA and lipid, mix gently and incubate 10–20 min at room temperature.
Add to cells
Add complexes dropwise to the well and rock gently.
Culture
Incubate at 37 °C. Change the medium after 4–6 h if cells are sensitive.
Analyse
Check expression 24–72 h later (fluorescence, western blot, qPCR).
Critical points
- Optimise the DNA : lipid ratio for each cell type.
- Plasmid quality (endotoxin, purity) strongly affects efficiency and toxicity.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Low efficiency | Optimise ratio and confluence, use fresh DNA, try another reagent or electroporation. |
| High toxicity | Reduce reagent amount, change medium after 4–6 h, increase cell density. |
Safety
Follow your institutional rules for recombinant DNA.
Further reading
- Manufacturer instructions for your transfection reagent.
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.