Cloning Ligation

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  • Molecular cloning is a method to prepare a recombinant DNA molecule, an extra-chromosomal circular DNA that can replicate autonomously within a microbial host. DNA ligation is commonly used in molecular cloning projects to physically join a DNA vector to a gene of interest. The ends of the DNA fragments can be blunt or cohesive and must contain monophosphate groups on the 5' ends. Following the mechanism described above, the covalent bonds are formed and a closed circular molecule is created that is capable of transforming a host bacterial strain. The recombinant plasmid maintained in the host is then available for amplification prior to downstream applications such as DNA sequencing, protein expression, or gene expression/functional analysis.

    Learn more about NEB's extensive selection of DNA Ligases

    DNA Ligase Selection Chart
    Tips for Maximizing Ligation Efficiencies
    Troubleshooting Guide for Ligases

    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. Are some ligations more difficult than others?

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

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

      The optimal reactant ratio is contingent upon the downstream application.

    4. What are the best conditions for DNA ligation?

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

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

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

    6. 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.

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    Protocols for Cloning Ligation