PEI transfection (HEK293T)
Cheap, efficient transfection for HEK293T (also good for HEK293, 293F). Use linear PEI MAX (Polysciences 24765, MW 40,000). About 1/100 the cost of Lipofectamine for similar yield in HEK293T.
PEI stock (1 mg/mL)
- Weigh 100 mg PEI MAX. Add to 90 mL endotoxin-free H₂O at room temp.
- Stir overnight to dissolve. Adjust to pH 7.0 with HCl (it'll start basic).
- Bring to 100 mL with H₂O. Filter through 0.22 µm.
- Aliquot 1 mL at −20 °C (long-term) or 4 °C for ~6 months (working aliquot).
Transfection (one 10 cm dish, ~5 µg DNA)
| Plate format | DNA | PEI (1 mg/mL) | OptiMEM (or DMEM) |
|---|---|---|---|
| 96-well | 0.1 µg | 0.3 µL | 10 µL |
| 24-well | 0.5 µg | 1.5 µL | 50 µL |
| 6-well | 2 µg | 6 µL | 200 µL |
| 10 cm dish | 10 µg | 30 µL | 1 mL |
| 15 cm dish | 25 µg | 75 µL | 2.5 mL |
Ratio: 3 µL PEI per 1 µg DNA (3:1 PEI:DNA mass ratio). Tweak between 2:1 and 4:1 to optimise.
Procedure (10 cm dish, day 0 = transfection day)
- Day −1: Plate 4 × 10⁶ HEK293T in 10 mL DMEM-10. Should be ~70% confluent at transfection.
- Day 0: In a sterile 1.5 mL tube:
- Mix 10 µg DNA + 1 mL OptiMEM (or DMEM, no FBS/antibiotic). Vortex briefly.
- Add 30 µL PEI (3:1 mass ratio). Vortex 5 s.
- Incubate at room temperature 15–20 min (don't exceed 30 min — complexes aggregate).
- Add the entire complex dropwise to the dish. Rock gently to distribute.
- Return to 37 °C, 5% CO₂.
- Optional: change media after 4–6 h to reduce PEI toxicity (boost viability).
- Harvest 24–72 h post-transfection depending on your readout.
Tips
- Quality of plasmid prep matters. Use endotoxin-free midi/maxi for high-yield transfections.
- For transient protein production, harvest at 48 h. For virus, see lentivirus production.
- Healthy log-phase cells transfect best. Avoid passages > 25.
- Don't use antibiotics in the transfection medium — they can be co-internalised and become cytotoxic.
- If you see cell death > 50% at 24 h, drop PEI:DNA ratio to 2:1.