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    DNA Ligation

    Ligation of DNA is a critical step in many modern molecular biology workflows. The sealing of nicks between adjacent residues of a single-strand break on a double-strand substrate and the joining of double-strand breaks are enzymatically catalyzed by DNA ligases. The formation of a phosphodiester bond between the 3' hydroxyl and 5' phosphate of adjacent DNA residues proceeds in three steps: Initially, the ligase is self-adenylated by reaction with free ATP. Next, the adenyl group is transferred to the 5'-phosphorylated end of the "donor" strand. Lastly, the formation of the phosphodiester bond proceeds after reaction of the adenylated donor end with the adjacent 3' hydroxyl acceptor and the release of AMP. In living organisms, DNA ligases are essential enzymes with critical roles in DNA replication and repair. In the lab, DNA ligation is performed for both cloning and non-cloning applications.

    1. DNA Ligation

      Ligation, the process of joining DNA fragments with a DNA ligase, proceeds in three steps. Learn more about ligation with our quick animation.

    2. How do I choose the best DNA Ligase?

      NEB continues to develop and produce the most extensive commercially available selection of high-quality, and performance-optimized DNA ligases and ligase master mixes for your ligation needs.

    3. What molar ratios should I use for DNA Ligation?

      The optimal reactant ratio is contingent upon the downstream application.

    4. Why do I need to add PEG to my DNA ligation?

      Polyethylene glycol (PEG) is an important reagent in ligation reactions, find out why.

    5. What are the best conditions for DNA ligation?

      Find out how the downstream application dictates the best reaction conditions for ligation.

    6. Are some ligations more difficult than others?

      Ligation of blunt ends and single-base overhangs require optimized reaction conditions.

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    DNA Ligation includes these areas of focus:

    Cloning Ligation
    Non-Cloning Ligation

    Protocols for DNA Ligation