* Ligation failed due to high salt or EDTA in the reaction. Clean up the DNA.
* CIP, BAP or SAP not completely inactivated from dephosphorylation step. Follow the recommended procedure to remove the phosphatase.
* Ligation produced only linear DNA because the DNA concentration was too high. Keep the total DNA concentration between 1-10 μg/ml.
* The ligated end was a single base overhang. Use up to 5 μl concentrated ligase at 16°C overnight.
* The insert and the plasmid do not have phosphates. Note: primers may not have phosphates leading to problems blunt end ligating into CIPed vectors. Order primers with phosphates or kinase the primers.
* Too much ligation mixture was added to the cells. Add between 1-5 μl to 50 μl competent cells.
* The insert was large and the conditions didn't allow circularization. Reduce the insert concentration and use concentrated ligase at 16°C overnight.
* The ligase was inactive. Test on lambda HindIII or other convenient substrate.
* The ligation mix contained PEG and was incubated overnight. Extended ligation with PEG causes a drop off in transformation efficiency. This could be due to the gradual production of large linear pieces of DNA that can inhibit transformation. The buffer for the Quick Ligation Kit (NEB# M2200) contains PEG.
* The ligation mix was not purified prior to electroporation. The buffer must be removed or a spark will be generated by the salt. Dialyse the sample or use a spin column to purify. The PEG in the Quick Ligation Kit Buffer (NEB# M2200 ) prevents sparking but it also prevents electroporation. PEG must be removed using a spin column.