Transfection Calculator — DNA & Reagent Volumes
Free transfection calculator. Lipofectamine 3000 amounts from the manufacturer table for 96-well to T-175, and area-based scaling of your own optimised condition for any reagent. Runs in your browser.
How it works
For Lipofectamine 3000 the calculator shows the manufacturer's own per-vessel amounts, from 96-well to T-175: growth medium, Opti-MEM, DNA, P3000 and the two lipid doses the protocol recommends testing. For any other reagent, you enter a condition that already works and it is scaled by growth-surface area, which keeps the DNA-to-reagent ratio and the amount per cm² constant, since transfection tracks complexes per area of monolayer rather than well volume.
Frequently asked questions
How much DNA do I need per well for a 24-well plate?
A 24-well plate well has about 2 cm² of growth area, and the usual working range is 0.5 to 1 µg of plasmid DNA per well for adherent lines. Scale from a condition you have already optimised rather than from a table: doubling the surface area doubles the DNA, the reagent and the medium together.
What DNA to lipid ratio should I use?
Ratios are reagent-specific and are given as volume of reagent per microgram of DNA. For Lipofectamine 3000 the manufacturer recommends testing two lipid doses, 1.5 and 3 µL per µg of DNA, with 2 µL of P3000 Reagent per µg of DNA. Use your own reagent's protocol for its ratio, and treat any published ratio as a starting point — the optimum shifts with cell line, confluence and plasmid size, so a small ratio titration is worth doing once per line.
Does this calculator account for cell type or transfection efficiency?
No, and that is deliberate. It scales volumes by growth surface area, which is geometry and holds for any cell type. Transfection efficiency depends on cell line, passage number, confluence and DNA quality, none of which can be derived from plate dimensions — so the tool scales what it can compute and leaves the biology to your own optimisation.
How do I scale a transfection from a 6-well plate to a 10 cm dish?
Scale by growth surface area, not by volume. A 6-well plate well is about 9.6 cm² and a 10 cm dish about 55 cm², so multiply DNA, transfection reagent and complex volume by roughly 5.7. Keep the DNA-to-reagent ratio constant — that ratio determines complex formation, and changing it is the usual reason a scaled-up transfection underperforms.
Why is my transfection efficiency low?
The most common causes are cell confluence outside the optimal 70–90% window, DNA of insufficient purity (a 260/280 ratio below 1.8 often indicates protein or phenol carry-over), antibiotics in the medium during complex formation, and an incorrect DNA-to-lipid ratio. Cell passage number matters too — efficiency commonly declines in high-passage cultures.