What is transformation efficiency?

It is the number of colony-forming units produced per microgram of plasmid DNA. It measures how readily a batch of competent cells takes up and expresses DNA, and it is the standard way to compare cell preparations and protocols.

Why scale by the volume plated?

You almost never plate the entire transformation. If you spread only a tenth of the recovery volume, the colonies you count represent a tenth of the DNA. The calculator multiplies the DNA mass by the plated fraction so the efficiency reflects the DNA actually on that plate.

What is a good efficiency?

Homemade chemically competent cells often reach 1e6 to 1e7 CFU per microgram, which is fine for routine cloning. Commercial high-efficiency cells reach 1e8 to 1e9 and are reserved for library construction or difficult ligations where every transformant counts.

Why count only 30 to 300 colonies?

Below 30 colonies counting error is large, and above 300 colonies start to merge and compete for nutrients, so you undercount. Plates outside this window give unreliable efficiency numbers. Dilute or plate a smaller fraction to land in range.

Should I use supercoiled plasmid to benchmark cells?

Yes. Efficiency depends heavily on the DNA. A pure, supercoiled control plasmid such as pUC19 gives the cleanest benchmark. A ligation mix transforms far less efficiently, so do not compare its numbers against a control-plasmid spec.

Bacterial Transformation Efficiency Calculator

Transformation efficiency tells you how good a batch of competent cells is and whether a transformation worked as expected. This calculator returns the standard figure, colony-forming units per microgram of DNA, correcting for the fact that you rarely plate the whole reaction.

How it works

The efficiency is colonies divided by the DNA mass on the plate. The DNA on the plate is the DNA used in the reaction scaled by the fraction of the recovery volume you actually spread:

DNA on plate (ug) = DNA used (ug) x (volume plated / total volume)
efficiency         = colonies / DNA on plate

For example, 250 colonies from a reaction using 0.1 ng of DNA, recovered in 1,000 µL with 100 µL plated, gives 0.0001 ug x (100 / 1000) = 0.00001 ug on the plate and 250 / 0.00001 = 2.5e7 CFU/ug.

Tips and notes

Always count a plate in the 30 to 300 colony range so counting error stays small and colonies do not merge. Benchmark cells with a pure supercoiled control plasmid rather than a ligation, because ligated DNA transforms far less efficiently and would understate cell quality. If efficiency drops over time, check that competent cells were kept at minus 80 degrees and thawed on ice, and that no heat-shock or recovery step was skipped.