Cloning: no colonies

1. Ranked causes

   1. Antibiotic plate wrong, expired, or too concentratedPositive-control plasmid also yields zero CFU on the same plate batch.
   2. Competent cells inactive (storage, thaw, or wrong strain)Same ligation succeeds in a fresh competent-cell aliquot.
   3. Ligation / assembly failed (ratios, insert:vector ratio, time)Uncut vector control gives colonies; ligation mix does not.
   4. Heat-shock / electroporation conditions offPositive-control DNA fails on this protocol but works with standard heat-shock timing.
   5. Recovery media / incubation temperature wrong after transformationSkipping 37 °C recovery or using antibiotic too early kills transformants.

2. Recovery steps

   1. Transform a known-good plasmid on the same competent cells and plate batchControls-first split between cells/plates vs ligation chemistry.
   2. Pour fresh antibiotic plates at the validated concentration; check expiryOver-strong or degraded antibiotic is a classic zero-colony cause.
   3. Re-ligate with a fresh insert:vector ratio and include a no-insert controlConfirms assembly chemistry before changing transformation parameters.
   4. Use standard heat-shock (42 °C / 30–45 s) and SOC recovery 1 h at 37 °CRecovers many protocol-drift failures.

3. Controls first

   • Did the positive-control plasmid yield colonies on the same cells and antibiotic plates?If no: Cells, plates, or transformation conditions — not the ligation alone.
   • Did the positive-control plasmid yield colonies on the same cells and antibiotic plates?If yes: Ligation/assembly or insert quality — keep cells/plates as working controls.

Diagnose your run

Opens Diagnostics capture with this first-party playbook behind it. Sign in if the workspace asks.