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  • 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