Cloning: low transformation efficiency

1. Ranked causes

   1. Competent cells partially compromised (age, freeze-thaw, warm thaw)Fresh aliquot restores CFU with the same positive-control DNA.
   2. Too much ligation mix / salts in the transformationDiluting ligation or column-cleaning DNA before transform raises CFU.
   3. Heat-shock timing or temperature offReturning to 42 °C / 30–45 s recovers efficiency.
   4. Antibiotic plates slightly over-strength (borderline selection)Lower antibiotic concentration increases CFU without raising empty-vector rate.

2. Recovery steps

   1. Thaw a fresh competent-cell aliquot on ice; avoid re-freezingPartial competence loss is the top efficiency drop cause.
   2. Use ≤10% ligation volume in the transformation or purify DNA firstExcess salts/enzymes suppress competence.
   3. Standardise heat-shock to 42 °C / 45 s with SOC recovery 1 hRecovers many protocol-drift efficiency losses.

3. Controls first

   • Is CFU also low for the positive-control plasmid on these competent cells?If yes: Cells, transformation conditions, or plates — not just this ligation.

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