Transfection Calculator

How to Use the Transfection Calculator

Select your vessel format, then choose a transfection reagent from the presets or enter a custom ratio. Enter the DNA amount per well and your DNA stock concentration. The calculator outputs exact volumes for tube A (DNA in Opti-MEM), tube B (reagent in Opti-MEM), the combined complex mix, and a step-by-step protocol.

DNA:Lipid Ratio Explained

The DNA:lipid ratio determines how efficiently nucleic acid is packaged into lipid nanoparticles. The ratio is expressed as µL of reagent per µg of DNA. Most manufacturers recommend a starting ratio of 2–3:1 (µL reagent per µg DNA), but optimal ratios vary by cell type and must be experimentally determined.

Key Tips for High Transfection Efficiency

• Seed cells 18–24 hours before transfection so they are 70–80% confluent at the time of transfection.
• Use serum-free or reduced serum media (Opti-MEM) for complex formation — serum inhibits lipid-DNA binding.
• Mix DNA and reagent separately in Opti-MEM before combining — this ensures uniform complex formation.
• Incubate complexes for exactly 10–20 minutes at room temperature — longer incubation leads to aggregate formation.
• Add complexes dropwise to cells in complete media; do not replace media unless specified by the protocol.
• Analyse results 24–72 hours post-transfection depending on the transgene expression level required.

Seeding Density for Transfection

Cells should be actively dividing and in exponential growth phase when transfected. Too sparse and cells will not form a monolayer; too confluent and cells become contact-inhibited, reducing uptake. Aim for 70–80% confluency at time of transfection.