- Ligation failed because there was no ATP or Mg2+. Use the supplied buffer or add ATP to a compatible buffer. ATP in buffers older than one year may have degraded enough to cause problems. When supplementing with ATP, be sure to use riboATP as deoxyriboATP will not work.
- Ligation failed due to EDTA in the reaction. Clean up the DNA (we recommend Monarch® nucleic acid purification kits).
- CIP, BAP, Antarctic Phosphatase or SAP are not completely inactivated following dephosphorylation step. Follow the recommended procedure to remove the phosphatase.
- Concentration was too high resulting in ligation only producing linear DNA. Keep the total DNA concentration between 1-10 μg/ml.
- The insert and the plasmid do not have phosphates. Note: primers may not have phosphates, preventing blunt-end ligation with vectors that have been treated with CIP. Order primers with phosphates or phosphorylate the primers with T4 Polynucleotide Kinase prior to use.
- 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.
- The ligase was inactive. Test on Lambda HindIII DNA 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. StickTogether™ DNA Ligase Buffer 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. Dialyze the sample or use a spin column to purify (we recommend Monarch nucleic acid purification kits). The PEG in StickTogether™ DNA Ligase Buffer prevents sparking but it also prevents electroporation. PEG must be removed using a spin column.