Cloning: satellite colonies

1. Ranked causes

   1. Antibiotic degraded or under-dosed in the plateFresh plates at correct concentration suppress satellites; old plates do not.
   2. Plate incubated too long (antibiotic locally depleted)Satellites appear after extended incubation; early pick avoids them.
   3. Ampicillin used when a more stable antibiotic is required for long incubationsSwitching to carbenicillin reduces satellites for the same vector backbone.
   4. Crowded plating — β-lactamase from true colonies rescues neighboursDiluting the transformation before plating reduces satellite rings.

2. Recovery steps

   1. Pour fresh plates at the validated antibiotic concentration; store cold and use promptlyDegraded antibiotic is the usual satellite driver.
   2. Pick large colonies early; avoid incubating amp plates overnight-plusLimits local antibiotic breakdown around true transformants.
   3. Dilute transformation before plating to reduce CFU densityFewer true colonies means less secreted β-lactamase rescue.

3. Controls first

   • Do satellite colonies also appear around the positive-control plasmid colonies on the same plate batch?If yes: Plate antibiotic / incubation — not a ligation-specific artefact.

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