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  • Tips for Maximizing Ligation Efficiencies

    NEB T4 DNA Ligase (NEB #M0202) is the most extensively used ligase for cloning-based experiments. Typically, a ligation reaction (blunt or cohesive ends) using traditional T4 DNA Ligase involves incubation at 16°C using 0.1-1 µM DNA (5´ termini) in 1X T4 DNA Ligase buffer. For your convenience, T4 DNA Ligase can also be used at room temperature, and is available in concentrated form (NEB #M0202). Alternatively, NEB’s Quick Ligation™ Kit (NEB #M2200) is uniquely formulated to ligate blunt or cohesive ends in 5 minutes at room temperature. The following tips will help to achieve maximum results from your ligation reactions.

    Reaction Buffers

    • T4 DNA Ligase Buffer (NEB #B0202) should be thawed on the bench or in the palm of your hand, and not at 37°C (to prevent breakdown of ATP).
    • Once thawed, T4 DNA Ligase Buffer should be placed on ice.
    • Ligations can be performed in any of the four standard restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer (NEB #B0201) if they are supplemented with 1 mM ATP.
    • When supplementing with ATP, use ribo ATP (NEB #P0756). Deoxyribo ATP will not work.
    • Before ligation, completely inactivate restriction enzyme by heat inactivation, spin column or Phenol/EtOH purification.


    • Use purified DNA preparations without EDTA or high salt concentrations.
    • Either heat inactivate (Antarctic Phosphatase) or remove phosphatase (CIP, BAP or rSAP) before ligation.
    • Keep total DNA concentration between 1-10 µg/ml.
    • Vector:Insert molar ratios between 1:1 and 1:10 are optimal for single insertions (up to 1:20 for short adaptors). Use NEBioCalculator to calculate molar ratios.
    • For cloning more than one insert, we recommend the Gibson Assembly Cloning Kit (NEB #E5510).
    • If you are unsure of your DNA concentration, perform multiple ligations with varying ratios.


    • For most ligations (blunt or cohesive) traditional T4 DNA Ligase or the Quick Ligation Kit are recommended.
    • For single base overhangs, it is recommended to use up to 5 µl concentrated ligase at 16°C overnight.
    • For large inserts, reduce insert concentration and use concentrated ligase at 16°C overnight.
    • T4 DNA Ligase can be heat inactivated at 65°C for 20 minutes.
    • Do not heat inactivate if there is PEG in the reaction buffer because transformation will be inhibited. The Quick Ligation Kit contains PEG.


    • Add between 1-5 µl of ligation mixture to competent cells for transformation.
    • Extended ligation with PEG causes a drop off in transformation efficiency (Quick Ligation Kit).
    • Electroporation is recommended for large constructs (>10,000 bp). Dialyze sample or use a spin column to purify first.

    Quick Ligation™ is a trademark of New England Biolabs, Inc.