How to Calculate Transformation Efficiency

The formula

Transformation efficiency is the number of colonies you would get per microgram of plasmid DNA if you plated everything:

efficiency (cfu/µg) = colonies × (total volume ÷ plated volume) × dilution factor ÷ µg of DNA

It is measured with a known amount of an uncut, supercoiled control plasmid such as pUC19, because the result depends on the plasmid as much as on the cells.

A worked example

You add 10 pg (0.00001 µg) of pUC19 to 50 µL of competent cells, add 950 µL of SOC after the heat shock (1,000 µL in total), and plate 100 µL of a 1:10 dilution. The next day there are 45 colonies.

1. Fraction plated: 100 ÷ 1,000 = 0.1, so multiply by 10.
2. Dilution: 1:10, so multiply by 10 again.
3. Colonies per transformation: 45 × 10 × 10 = 4,500.
4. Per µg: 4,500 ÷ 0.00001 = 4.5 × 10⁸ cfu/µg.

What is a good number?

• Home-made chemically competent cells: 10⁶ to 10⁷ cfu/µg.
• Commercial chemically competent cells: 10⁸ to 10⁹.
• Electrocompetent cells: 10⁹ to 10¹⁰.

A ligation gives far fewer colonies per µg than the control plasmid, often a hundred to a thousand times fewer, because ligated DNA is mostly linear or relaxed and the ligation buffer inhibits transformation.

Why efficiency drops

Too much DNA (more than about 100 ng per 50 µL of cells) saturates the cells and lowers the per-µg number. Salt, ethanol or ligase buffer in the DNA, cells that warmed up before the heat shock, thawed and refrozen cells, and skipping the recovery step all cut efficiency. Large plasmids transform less well than small ones.

Calculators

The Transformation Efficiency Calculator does the arithmetic from colonies, DNA, volumes and dilution. Check the DNA amount with the DNA Concentration Calculator, and turn nanograms into molecules with the DNA Copy Number Calculator.