Show MeA step-by-step polyethylenimine (PEI) transfection protocol forms DNA–PEI polyplexes in serum-free medium, lets them mature for 15–30 minutes, and adds them to cells at 60–80% confluency using 2–3 µL of 1 mg/mL linear 25 kDa PEI per 1 µg DNA. Replace medium with PEI-containing mix for 4–6 hours, then restore complete medium. Reporter expression typically peaks at 24–48 hours. At a DNA:PEI mass ratio near 1:3, HEK293T cells commonly reach 50–80% transfection efficiency with minimal cytotoxicity.
How PEI Transfection Works at the Molecular Level
Polyethylenimine is a cationic polymer dense in protonatable amine groups. Roughly 25% of its nitrogen atoms are protonated at physiological pH, which leaves the rest available to bind protons inside the endosome. This buffering capacity is the whole trick of PEI.
When DNA and PEI are mixed, the positively charged polymer collapses the negatively charged plasmid into a compact particle 50–150 nm across. Cells internalize these polyplexes mainly through clathrin-mediated endocytosis and, for larger aggregates, macropinocytosis. Once inside the endosome, PEI soaks up protons pumped in by the v-ATPase. The endosome cannot acidify efficiently, so the proton sponge effect draws in chloride ions and water, osmotically swelling and rupturing the vesicle before lysosomal enzymes destroy the DNA.
In plain terms: PEI acts as a molecular sponge that makes the cellular recycling compartment burst before it can digest the cargo. The DNA then escapes into the cytoplasm and enters the nucleus during mitosis, which is why dividing cells transfect far better than quiescent ones.
Two structural facts matter. Linear 25 kDa PEI outperforms branched PEI for DNA delivery in most suspension and adherent lines, while branched PEI is favored for siRNA. The 25 kDa size sits near a sweet spot: smaller PEIs (under 2 kDa) form unstable complexes, and much larger ones raise cytotoxicity sharply.
Why the DNA-to-PEI Ratio Drives Everything
The mass ratio of DNA to PEI controls charge balance, particle size, and toxicity in a tight window. Too little PEI leaves the polyplex undercharged, so it cannot bind the anionic cell surface. Too much PEI produces free polymer that perforates membranes and kills cells.
At a 1:3 mass ratio, the nitrogen-to-phosphate (N/P) ratio lands near 10, which is the most widely cited optimum for linear 25 kDa PEI in HEK293T cells. Pushing to 1:6 raises efficiency slightly in some lines but typically doubles cytotoxicity measured by metabolic assays. Dropping to 1:1 cuts efficiency roughly in half while only modestly improving viability.
The efficiency figure you get depends heavily on the assay. Flow cytometry of a GFP reporter and luciferase luminometry often disagree by 20–40% on the same samples, because they measure different things: the fraction of cells expressing versus total protein output. Report both if the number matters.
The Full PEI Transfection Protocol Timeline
The table below gives the working schedule for a standard 6-well plate format with HEK293T cells. Scale reagent volumes linearly with surface area; a 10 cm dish takes roughly 5 times the DNA and PEI of one 6-well well.
| Time point | Step | Key parameters |
|---|---|---|
| Day −1 (16–24 h before) | Seed cells | 2–3 × 10^5 cells per 6-well well; target 60–80% confluency at transfection |
| Day 0, t = 0 min | Dilute DNA and PEI separately | 2 µg DNA in 100 µL Opti-MEM; 6 µL PEI (1 mg/mL) in 100 µL Opti-MEM |
| Day 0, t = 5 min | Combine and mix | Add PEI to DNA dropwise; vortex 5 s; incubate 15–30 min at room temperature |
| Day 0, t = 30 min | Add polyplexes | Apply 200 µL mix per well; swirl gently |
| Day 0, t = 4–6 h | Medium exchange | Replace with complete medium; optional for HEK293T, near-mandatory for sensitive lines |
| Day 1 (24 h) | First assay window | Reporter expression detectable; viability check |
| Day 2 (48 h) | Peak expression | Optimal harvest for most transient assays |
| Day 3 (72 h) | Decline phase | Expression falls 30–60% from peak; do not assay past this without reason |
The 15–30 minute complexation window is not arbitrary. Polyplexes smaller than about 15 minutes of maturation are loosely formed and dissociate at the cell surface; beyond 60 minutes they aggregate into particles above 1 µm that sediment and cause focal toxicity.
Bench-Scale Variations by Format and Cell Line
Volumes scale with growth area, but the per-area dose should stay constant. The table compares common formats using the same 1:3 ratio and 1 µg DNA per cm² of growth area, a moderate dose suitable for most lines.
| Format | Growth area | DNA per well | PEI (1 mg/mL) | Total polyplex volume |
|---|---|---|---|---|
| 96-well | 0.32 cm² | 0.1 µg | 0.3 µL | 10 µL |
| 24-well | 1.9 cm² | 0.5 µg | 1.5 µL | 50 µL |
| 6-well | 9.6 cm² | 2 µg | 6 µL | 200 µL |
| 10 cm dish | 55 cm² | 10 µg | 30 µL | 1 mL |
| T-75 flask | 75 cm² | 15 µg | 45 µL | 1.5 mL |
Cell line choice shifts the optimum more than format does. HEK293T tolerates serum during complexation and needs no medium exchange. CHO cells prefer a 1:2 ratio and benefit from a 4-hour exchange. HeLa and NIH/3T3 are more sensitive to free PEI, so a 1:2.5 ratio with a 5-hour exchange reduces death. Primary neurons and many stem cells resist PEI entirely and need Lipofectamine or electroporation instead.
Suspension-adapted HEK293 cells require a dedicated variant: complex in full medium at 1–2 × 10^6 cells/mL and skip the exchange step, since centrifugation to change medium damages suspension cultures more than the residual PEI does.
Common Mistakes and Corrected Misconceptions
The most frequent error is mixing DNA and PEI in medium that contains serum or antibiotics. Serum proteins compete for polyplex binding, and this can cut efficiency by half. Always dilute both components in reduced-serum medium such as Opti-MEM.
A widespread misconception holds that more PEI means more transfection. Beyond a 1:6 ratio, free polymer dominates and metabolic assays show viability dropping below 70% within 48 hours, which erases any efficiency gain. The dose-response curve is a plateau, not a ramp.
Another corrected belief: DNA quality is not a minor detail. Endotoxin-contaminated miniprep DNA can suppress transfection 5- to 10-fold. Use endotoxin-free maxiprep plasmid at a 260/280 ratio of 1.8–1.9.
Watch for these red flags in a failing prep:
- Efficiency below 20% in a previously working line, suggesting degraded PEI stock older than 6 months.
- Uniformly dead wells at 24 hours, indicating a ratio error above 1:6.
- Patchy, edge-biased expression, indicating uneven plate swirling or evaporation.
- Visible precipitate in the complexation tube, indicating aggregation from over-long incubation.
- No signal at all with a positive-control plasmid, pointing to a DNA or handling fault, not the PEI.
What to Actually Do: A Reliable Working Sequence
Follow this ordered sequence rather than improvising, and record every variable.
- Seed cells the day before at a density that lands at 60–80% confluency on transfection day; test your line's doubling time once and calculate rather than guess.
- Warm Opti-MEM, DNA, and PEI separately to room temperature; cold reagents slow complexation.
- Dilute DNA in one tube and PEI in another, each in half the final volume, then add PEI to DNA dropwise while vortexing.
- Incubate exactly 20 minutes, then apply the mix without pipetting up and down against the cell monolayer.
- For sensitive lines, exchange to complete medium at 4–6 hours; for HEK293T, exchange is optional but improves viability.
- Run a positive-control GFP plasmid alongside every batch, and assay at 24 and 48 hours before choosing a harvest time.
Keep a single PEI stock aliquot at 4 °C protected from light; repeated freeze-thaw cycles degrade it and shift your ratio. Prepare fresh working dilutions weekly.
Limitations and When to Choose Another Method
PEI transfection is cheap, scalable, and effective in HEK293 and CHO lines, but it fails in hard-to-transfect cells and cannot deliver to primary or non-dividing cells efficiently because nuclear entry depends on mitosis. It also triggers innate immune responses in some lines and is unsuitable for in vivo work without modification.
For difficult cells, lipid reagents achieve higher efficiency at higher cost, and electroporation or viral vectors suit primary and in vivo applications. Stability and reproducibility, not peak efficiency, are PEI's real advantages at scale.