Troubleshooting Tips for Ligation Reactions
- Add controls, including vector alone, insert alone and uncut vector.
- Vary the molar ratio from 1:1 to 1:10 vector:insert (1:20 for short adaptors). Use NEBioCalculator for molar ratio calculations.
- Insert or plasmid should have a 5´ phosphate.
- Use fresh buffer as the ATP or DTT may degrade over time.
- Total DNA concentration should be less than 10 ng/μl.
- When excising a fragment from a gel, minimize UV exposure and use longwave UV (360 nM) to reduce damage to the DNA