Transfection Mix Calculator (DNA, Reagent, Medium per Well)

How it works

DNA total = µg per well × wells × (1 + overage) · reagent = DNA × ratio (µL/µg) · stock volume = DNA ÷ stock concentration · medium per tube = per-well volume × wells × (1 + overage)

Cationic lipid and polymer reagents wrap negatively charged plasmid DNA into complexes that cells take up, and the amount of complex a culture tolerates scales with the well's surface area — which is why every reagent manual gives a per-format table. The reagent-to-DNA ratio controls the charge balance of the complex: too little reagent and the DNA is not packaged, too much and the lipid is toxic. Preparing the DNA and reagent in separate tubes of serum-free medium before combining them keeps serum proteins from coating either component prematurely, and making one master mix with a small overage guarantees every well receives an identical complex despite pipetting losses.

Worked example

Six wells of a 6-well plate at the standard 2.5 µg DNA and 3 µL reagent per µg, with 10% overage (6.6 well-equivalents): 16.5 µg plasmid — 16.5 µL of a 1 µg/µL stock — into 825 µL medium, and 49.5 µL reagent into another 825 µL. Combine, wait 15–20 minutes, and add 250 µL of complex to each well seeded the day before with 2.5–5 × 10⁵ cells.

Frequently asked questions