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Troubleshooting Guide for Ligases

Problem Cause Solution
Few or no transformants
 Too much ligation mixture was used
  • Use < 5 μl of the ligation reaction for the transformation
Inefficient ligation
  • Make sure that at least one fragment being ligated contains a 5´ phosphate moiety
  • Vary the molar ratio of vector to insert from 1:1 to 1:10 (1:20 for short adaptors). Use NEBioCalculator to calculate molar ratios.
  • Purify the DNA to remove contaminants such as salt and EDTA
  • ATP will degrade after multiple freeze-thaws; repeat the ligation with fresh buffer
  • Heat inactivate or remove the phosphatase prior to ligation
  • Ligation of single base-pair overhangs (most difficult) may benefit from being carried out with Blunt/TA Master Mix (NEB #M0367), Quick Ligation™ Kit (NEB #M2200) or concentrated T4 DNA Ligase (NEB #M0202)
  • Test the activity of the ligase by carrying out a ligation control with Lambda-HindIII digested DNA (NEB #N3012)
Ran the ligation on a gel and saw no ligated product Inefficient ligation
  • Make sure at least one DNA fragment being ligated contains a 5´ phosphate
  • Vary the molar ratios of vector to insert from 1:1 to 1:10  (1:20 for short adaptors). Use NEBioCalculator to calculate molar ratios.
  • Purify the DNA to remove contaminants such as salt and EDTA
  • ATP will degrade after multiple freeze-thaws; repeat the ligation with fresh buffer
  • Heat inactivate or remove the phosphatase prior to ligation
  • Ligation of single base-pair overhangs (most difficult) may benefit from being carried out with Blunt/TA Master Mix (NEB #M0367), Quick Ligation Kit (NEB #M2200) or concentrated T4 DNA Ligase (NEB #M0202)
  • Test the activity of the ligase by carrying out a ligation control with Lambda-HindIII digested DNA (NEB #N3012)
The ligated DNA ran as a smear on an agarose gel The ligase is bound to the substrate DNA
  • Treat the ligation reaction with Proteinase K (NEB #P8107) prior to running on a gel