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 Ligation

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

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    • What is a Difficult Ligation?

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

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    • What are the Best Ratios of Reactants?

      The optimal reactant ratio is contingent upon the downstream application.

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    • What are the Best Reaction Times and Temperatures?

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

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    • Why Use PEG in a Ligation?

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

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    • Which Ligase Should I Use?

      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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