Transfection Calculator
How to use: pick an application preset below — it fills the DNA amount, reagent, and ratios for you. Then choose your vessel and how many, and read the bench recipe on the right. The advanced fields (N:P ratio, stock conc.) only need changing if you are optimizing.
Application Preset ?
Vessel Format
Number of Vessels
Total DNA (µg/vessel) ?
Transfection Reagent
2.50
µg DNA / vessel
Total DNA per vessel
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Reagent / vessel
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Complex / vessel
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DNA / 10⁶ cells
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Total DNA (1 vessel)
How it's calculated
Scale Comparison
Default DNA for each vessel at your current reagent ratio. Your own DNA entry is used only in the cards above.
Protocol Timeline

About Transfection Calculations

Transfection is the process of introducing exogenous nucleic acids into eukaryotic cells. The amount of DNA and transfection reagent required depends on the vessel format, cell line, and reagent system. This calculator supports the most widely used chemical transfection methods: polyethylenimine (PEI), cationic lipid reagents (Lipofectamine 2000, Lipofectamine 3000, FuGENE HD), and calcium phosphate co-precipitation. Each method forms DNA-reagent complexes that are taken up by cells via endocytosis.

For PEI-based transfection, the critical parameter is the nitrogen-to-phosphate (N:P) ratio, which describes the stoichiometric balance between protonatable amines in PEI and phosphate groups in DNA. The formula is: PEI mass = DNA mass x (N/P) x (mass per nitrogen / 330), where 330 Da is the average molecular weight per nucleotide of double-stranded DNA. For free-base PEI the mass per nitrogen is 43.07 Da (one CH₂CH₂NH repeat unit); PEI MAX is the hydrochloride salt (MW 40,000, about 22,000 free base), so it is about 78.3 Da. Optimal N:P ratios are typically about 8 to 13 (a 2:1 to 3:1 PEI:DNA mass ratio) for PEI MAX, around 23 to 38 (3:1 to 5:1) for 25 kDa linear PEI, and 6 to 10 for branched 25 kDa PEI.

Co-transfection with multiple plasmids is routine in viral vector production. AAV manufacturing uses triple transfection with pHelper, pRC (Rep/Cap), and pAAV-GOI (transgene) plasmids, typically at a 1:1:1 mass ratio. Lentiviral vector production uses third-generation packaging with four plasmids: transfer vector, pMDLg/pRRE (Gag-Pol), pRSV-Rev, and pMD2.G (VSV-G envelope), at approximately 4:2:1:1 mass ratios. This calculator supports both mass and molar ratio modes, automatically converting between them based on plasmid sizes.

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Frequently Asked Questions

How much DNA should I use per well for transfection?

DNA amount scales with vessel surface area. Typical starting amounts: 96-well 0.1 µg, 24-well 0.5 µg, 12-well 1 µg, 6-well 2.5 µg, 100 mm dish 10 µg, T-75 flask 15 µg. For suspension cultures, use 1 µg/mL of culture volume. Optimize by testing 0.5x to 2x of the recommended amount.

How much DNA per well should I use for a 6-well plate transfection?

For adherent HEK293, CHO, or HeLa cells at ~70-90% confluence in a 6-well plate (9.6 cm² surface area, 2 mL medium), the standard starting DNA amount is ~2.5 µg per well. This scales linearly with vessel surface area: 0.1 µg for 96-well, 0.5 µg for 24-well, 1 µg for 12-well, 2.5 µg for 6-well, 10 µg for a 100 mm dish. For suspension cultures (HEK293T, ExpiCHO) the target is ~1 µg DNA per mL of culture at 1-2 million cells/mL. Optimise between 0.5x and 2x of these starting values and keep the reagent-to-DNA ratio constant across the sweep.

What is the typical PEI to DNA ratio for HEK293 transfection?

For HEK293 and HEK293T with PEI MAX (linear 40 kDa), the standard PEI:DNA mass ratio is 2:1 to 3:1 (N:P about 8-13, because PEI MAX is the hydrochloride salt at about 78 g/mol per nitrogen). For 25 kDa linear PEI, use a slightly higher 3:1 to 5:1 mass ratio (N:P 23-38). Higher ratios give more complex compaction and better uptake but raise cytotoxicity, so most large-scale HEK293T suspension protocols (viral vector production) sit at 2:1 to 3:1 PEI MAX:DNA (w/w). Branched 25 kDa PEI runs at a lower N:P of 6-10 (~1:1 to 1.5:1 mass ratio). Titrate the ratio in a small pilot when switching plasmid or cell line.

What is the N:P ratio and how do I calculate it?

The N:P ratio is the molar ratio of nitrogen atoms in PEI to phosphate groups in DNA. For free-base PEI (linear or branched 25 kDa): N/P = (PEI mass / 43.07) / (DNA mass / 330), so PEI (µg) = DNA (µg) × N/P × 0.1305. PEI MAX is the hydrochloride salt (MW 40,000, about 22,000 free base), so each nitrogen carries about 78.3 Da and PEI MAX (µg) = DNA (µg) × N/P × 0.237. Optimal N:P is about 8-13 (a 2:1 to 3:1 PEI:DNA mass ratio) for PEI MAX, 23-38 (3:1 to 5:1) for 25 kDa linear PEI, and 6-10 for branched PEI.

Which transfection reagent should I use for my cell line?

PEI MAX is cost-effective and standard for large-scale HEK293T (viral vectors). Lipofectamine 2000 works well for most adherent lines (HEK293, HeLa, COS-7). Lipofectamine 3000 is best for hard-to-transfect and primary cells. FuGENE HD offers low toxicity for CHO, Jurkat, and sensitive lines. Calcium phosphate is inexpensive for HEK293 but less consistent.

What plasmid ratios should I use for AAV triple transfection?

The standard mass ratio is 1:1:1 for pHelper : pRC (Rep/Cap) : pAAV-GOI. Some protocols use 2:1:1 with extra helper. For equimolar ratios, adjust mass by plasmid size (pHelper ~11.6 kb, pRC ~7.4 kb, pAAV ~5 kb). Total DNA is typically 1 µg/mL suspension or 10 µg per 100 mm dish with PEI MAX at a 2:1 to 3:1 PEI:DNA mass ratio (N:P about 8-13).

How do I scale transfection from plates to bioreactors?

For adherent cultures, scale DNA proportionally to surface area. For suspension, scale by culture volume (1 µg/mL). Keep the reagent:DNA ratio constant. At bioreactor scale, use PEI for cost-effectiveness, prepare complexes in 10% of culture volume, and ensure rapid mixing. Maintain 1-2 × 10⁶ cells/mL at transfection.

When should I change media after transfection?

PEI: 4-6 hours post-transfection (optional for HEK293T suspension). Lipofectamine 2000: 4-6 hours (recommended to reduce toxicity). Lipofectamine 3000 and FuGENE HD: often not required due to lower toxicity. Calcium phosphate: 6-16 hours to remove precipitate. Harvest depends on product: secreted proteins 48-72h, AAV 48-96h, lentivirus 48-72h.

Further reading