Cell culture
Transfection setup
Scale a transfection from a per-well DNA amount and a reagent-to-DNA ratio across a whole plate, with an overage so the master mix covers pipetting losses. The tool does the arithmetic; the DNA amount and ratio must come from your reagent’s protocol and your own optimisation.
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Master mix
| Component | Per well | Total with overage |
|---|
The ratio and DNA amount depend entirely on your reagent and cell line — take them from the reagent’s protocol. This tool does the arithmetic; it does not know the right numbers for your experiment.
DNA per well is either entered directly or derived from an amount per square centimetre times the vessel’s growth area. Reagent volume is the DNA times the ratio you enter. Totals carry the overage so the master mix covers pipetting losses across all wells. Reduced-serum medium for complex formation is optional and scales the same way.
Formula
reagent per well = DNA per well × ratio; total = per well × wells × (1 + overage)
- DNA per well
- entered directly, or as an amount per cm² times the vessel’s growth area
- ratio
- microlitres of reagent per microgram of DNA, from the reagent datasheet
- overage
- a percentage added so the mix does not run short across all the wells
Worked example
Half a microgram per well at a 3:1 ratio across 24 wells
- 0.5 µg DNA per well
- ratio 3 µl/µg
- 24 wells, 10 per-cent overage
- Reagent per well: 0.5 times 3 is 1.5 µl.
- Working factor: 24 times 1.10 is 26.4 wells-worth.
- Totals: 13.2 µg DNA and 39.6 µl reagent.
Answer 1.5 µl reagent per well; 13.2 µg and 39.6 µl for the plate
Common mistakes
- Borrowing a ratio from a different reagent. Lipid reagents, PEI and others differ severalfold; the right ratio is the one on your product’s sheet, refined for your cells.
- Skipping the overage on small wells. Dead volumes and tip retention bite hardest at 96-well scale; without a margin the last wells come up short.
- Forgetting that confluence drives efficiency. The calculator sizes the mix, not the biology; seed to the density your protocol assumes.
Frequently asked
Micrograms per well or nanograms per cm²?
Per well is convenient for standard plates. Per cm² travels better between vessel formats, since it scales with growth area automatically.
Does this cover PEI by nitrogen-to-phosphate ratio?
No. It uses the volume-to-mass ratio that lipid and many polymer reagents are specified by. An N:P calculation needs different inputs and is out of scope here.
What overage should I use?
Ten per cent is a common starting point; go higher for many small wells or a multichannel step, lower for a few large ones.