Transformation efficiency calculator
Transformation efficiency is colony-forming units per microgram of DNA: CFU/µg = colonies × dilution × (recovery volume / plated volume) ÷ µg of DNA. Plating 100 µl of a 1 ml recovery from 1 ng of plasmid and counting 250 colonies gives 2.5 × 10⁶ CFU/µg.
Result
CFU/µg = colonies × dilution × (recovery vol / plated vol) / µg DNA. Measure with a supercoiled control plasmid (e.g. pUC19) — ligation products transform 10–100× worse than supercoiled DNA.
Doing this for a real construct?
Talindrew runs it on your actual sequence — editor, cloning wizards and analysis in the browser, free.
Formula
CFU/µg = colonies × dilution × (V_recovery / V_plated) / µg DNA
Worked example
Given: 1 ng pUC19 into cells, 1 ml recovery, 100 µl plated, 250 colonies, no extra dilution
- 1.Fraction plated: 100 / 1,000 = 0.1 → DNA represented on the plate = 0.1 ng = 10⁻⁴ µg.
- 2.CFU/µg = 250 / 10⁻⁴ = 2.5 × 10⁶.
Efficiency = 2.5 × 10⁶ CFU/µg
How the calculation flows
Units & constants
| Chemically competent (typical) | 10⁶–10⁸ CFU/µg |
|---|---|
| Commercial high-efficiency | 1–5 × 10⁹ CFU/µg |
| Electroporation | up to ~10¹⁰ CFU/µg |
| Cloning needs | ≥ 10⁶ for routine subcloning; ≥ 10⁸–10⁹ for libraries |
| Standard test DNA | Supercoiled pUC19, 10–100 pg |
| Price | Free |
Why measure with supercoiled plasmid, not your ligation?
Efficiency is a property of the competent cells, and supercoiled monomeric plasmid is the standard substrate: ligation products — nicked, linear, concatemeric — transform 10-to-100-fold worse. Measuring with pUC19 tells you whether the cells are good; a low colony count from a ligation with good cells tells you the ligation is the problem. Keeping the two diagnoses separate is the point of the number.
What kills transformation efficiency?
In rough order of impact:
- Warm handling — competent cells lose an order of magnitude sitting on wet ice too long or thawing warm.
- Too much DNA or too much ligation mix (salt and PEG carryover inhibit; keep ≤ 5 µl per 50 µl cells).
- Skipped or shortened recovery incubation before plating on selective media (especially for ampicillin alternatives).
- Freeze–thaw cycles of the competent cell aliquot.
Frequently asked questions
- Is 250 colonies from 1 ng good?
- That computes to 2.5 × 10⁶ CFU/µg if you plated a tenth of the recovery — fine for routine subcloning, far too low for library construction, which needs 10⁸–10⁹. Commercial high-efficiency cells plated the same way would give thousands of colonies from 10 pg.
- Why does the calculator ask for recovery and plated volumes?
- Because you rarely plate the whole transformation. Counting 250 colonies from a tenth of the mix means the whole transformation carried ~2,500 — the volumes scale the count back to the full reaction before dividing by DNA input.
Related calculators & guides
Use this calculator inside the app.
Free to use. No download required. Sign in and you land right back on this calculator.