Transfection Calculator
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 Vol (µL)
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Complex Vol (µL)
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DNA / 10⁶ cells
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Total (N vessels)
Scale Comparison
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 (43.07 / 330), where 43.07 Da is the molecular weight per nitrogen in the PEI repeat unit (CH₂CH₂NH) and 330 Da is the average molecular weight per nucleotide of double-stranded DNA. Optimal N:P ratios are typically 3 to 7 for linear PEI (PEI MAX) and 5 to 15 for branched 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.

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. Formula: N/P = (PEI mass / 43.07) / (DNA mass / 330). To calculate PEI mass from a target N:P: PEI (µg) = DNA (µg) × N/P × 0.1305. Optimal N:P is 3-7 for linear PEI and 5-15 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 N:P 5-7.

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.