• My NEB
  • Print
  • PDF
  • DNA Ligases

    Learn how cloning DNA ligases from NEB is a natural fit

    NEB’s enzymology expertise sets it apart from competitors, allowing us to produce enzymes for molecular biology that deliver; our highly pure enzymes, over 250 of which are recombinant, offer exceptional performance and value.

    A few of our more popular standalone ligases and ligase kits available are:


    • 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.

      scroll to see additional videos
    • What is a Difficult Ligation?

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

      scroll to see additional videos
    • Why Use PEG in a Ligation?

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

      scroll to see additional videos
    • 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.

      scroll to see additional videos
    • What are the Best Reaction Times and Temperatures?

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

      scroll to see additional videos
    • What are the Best Ratios of Reactants?

      The optimal reactant ratio is contingent upon the downstream application.

      scroll to see additional videos

    For added convenience and accuracy in your ligation reaction setup, try our new DNA Ligase Master Mixes. These pre-mixed, ready-to-use formulations include a proprietary ligation reaction enhancement agent for improved performance.

    For your convenience, master mixes are available for every end type:

    • For sticky ends, try our Instant Sticky-end Ligase Master Mix (NEB #M0370)
    • For blunt or single-base overhang ends, try our Blunt/TA Ligase Master Mix (NEB #M0367)

    For the full list of DNA Ligases and Ligase Master Mixes offered by NEB, as well as suggested applications, visit the DNA Ligase Selection Chart.

    For additional help, visit our Ligase Troubleshooting Guide.

    Extreme purity with NEB's T4 DNA Ligase

    Equivalent amounts of protein were loaded and silver stained using SilverXpress™. Marker M is NEB’s Broad Range Protein Marker (NEB #P7702).

    Blunt/TA Ligase Master Mix Outperforms the Competition

    Duplicate ligation reactions of blunt or T/A vector/insert pairs were set upaccording to the master mix vendors' suggestions. Equal amounts of ligated DNA were used to transform NEB 10-beta Competent E. coli (NEB #C3019) and triplicate plating was performed. Transformation results were averaged and graphed as a percentage of the highest performing reaction, T/A ligation using the Blunt/TA Ligase Master Mix.

    Instant Sticky-end Ligase Master Mix Offers Robust Ligation with No Incubation Step

    Reactions containing 20 ng of pUC19 digested with SacI/SphI, and a 3-foldmolar excess of a 1.6 kb fragment from a different plasmid with compatible ends were set up using the Instant Sticky-end Ligase Master Mix. Without any additional incubation time, 2 µl were immediately withdrawn and used to transform a 50 µl aliquot of NEB 10-beta Competent E. coli (NEB #C3019). A 50 µl aliquot of the 1 ml outgrowth was plated onto a selective plate and incubated overnight at 37°C. Results show that several hundred colonies wereproduced.

    ElectroLigase reactions are complete in 60 minutes or less

    Ligation reactions containing equal amounts (20 ng vector and 3-fold molar excess of insert) of blunt (A) or T/A (B)
    vector/insert pairs were set up using ElectroLigase and incubated for the times shown. After heat inactivation of the
    ligase, 2 μl of each reaction were withdrawn and directly used to transform NEB 10-beta Electrocompetent E. coli
    (NEB #C3020). 50 μl aliquots of the outgrowth (diluted, in some cases) was plated onto selective plates and incubated
    overnight at 37°C. Colonies were counted, adjusted for plating dilution, and graphed.

    Electroligase® is a registered trademark of New England Biolabs.
    Quick Ligation™ is a trademark of New England Biolabs
    SilverXpress™is a trademark of Life Technologies, Inc.

    Advantages

    • Highly pure enzyme with no lot-to-lot variation
    • Convenient formats include stand-alone enzyme or Quick Ligation Kit format
    • Flexible reaction setup
    • Active in a variety or reaction buffers

    Benefits of DNA Ligases and Ligase Master Mixes

    • Fast ligations
    • Robust ligation efficiency
    • Industry standard for purity
    • Most extensive selection commercially available 
    • New formulations optimized for your substrates 
    • Convenient master mix formats

    Quality Controls for T4 DNA Ligase

    Exonuclease contamination in a variety of T4 DNA Ligase samples as detected by capillary electrophoresis.

    Quality Controls for DNA Ligase
    Physical Purity Determined by SDS-PAGE analysis (or silver stained gel).
    Exonuclease Activity Contaminating exonucleases are assayed by monitoring release of radioactivity labeled DNA substrate. Very low levels of specific types of exonuclease contamination are also assayed for using capillary electrophoresis with dye labeled substrate.
    Nuclease Activity Contaminating nucleases are assayed by incubation with HindIII fragments of lambda DNA.
    Endonuclease Activity Incubation with supercoiled plasmid DNA for 4 hours in used to detect any nicking or non-specific nuclease degradation.
    Functional Assay Room temperature ligation is performed.

    Capillary electrophoresis testing of T4 DNA Ligase and other commercially available ligases. Reactions contained 20 nM fluorescent labeled substrate, 1X T4 Ligase Buffer, and 5 µl enzyme in a total reaction volume of 50 µl. Reactions were incubated at 37°C for 16 hours followed by heat inactivation at 80°C for 20 minutes. 1 µl of each reaction was combined with 10 µl of Hi Di™ Formamide containing GeneScan™ 120 LIZ® Size Standards and injected on the 3130 x 1 Genetic Analyzer. Three double-stranded substrates were tested and the data indicates whether the degradation occurs on the top or bottom strand.

    Hi Di™ is a trademark of Applera Corporation.
    GeneScan™ is a trademark of Life Technologies, Inc.
    120 LIZ® is a registered trademark of Life Technologies, Inc.

    Capillary Electrophoesis Testing of T4 DNA Ligase

    Exonuclease contamination in a variety of T4 DNA Ligase samples as detected by capillary electrophoresis.

    Capillary electrophoresis testing of T4 DNA Ligase and other commercially available ligases. Reactions contained 20 nM fluorescent labeled substrate, 1X T4 Ligase Buffer, and 5 µl enzyme in a total reaction volume of 50 µl. Reactions were incubated at 37°C for 16 hours followed by heat inactivation at 80°C for 20 minutes. 1 µl of each reaction was combined with 10 µl of Hi Di™ Formamide containing GeneScan™ 120 LIZ® Size Standards and injected on the 3130 x 1 Genetic Analyzer. Three double-stranded substrates were tested and the data indicates whether the degradation occurs on the top or bottom strand.

    Hi Di™ is a trademark of Applera Corporation.
    GeneScan™ is a trademark of Life Technologies, Inc.
    120 LIZ® is a registered trademark of Life Technologies, Inc.

    Reported Activities and Applications for T4 Ligases

    Relative Efficiency of T4 Ligases on Nicked Substrates

    Sticky-end Specific Ligation with T7 DNA Ligase

    Ligation reactions containing blunt- (ΦX174 DNA-HaeIII Digest, NEB #N3026) and sticky-end (λ- HindIII Digest, NEB #N3012) fragments were set up with 200 ng of each substrate and 1 µl of each ligase, and incubated for 30 minutes at 25°C in their corresponding reaction buffers. Reactions were immediately stopped with 6X loading dye and resolved by electrophoresis on a 1% agarose gel and stained with ethidium bromide.

    ElectroLigase® Reactions are Complete After 60 Minutes or Less, Even on Blunt- or T/A Ends

    Ligation reactions containing equal amounts (20 ng vector and 3-fold molar excess of insert) of blunt- (A), T/A (B), or sticky-end (C) vector/insert pairs were set up using ElectroLigase® and incubated for the times shown. After heat inactivation, 2 μl of each reaction were withdrawn and directly used to transform NEB 10-beta Electrocompetent E. coli (NEB #C3020). 50 μl aliquots of the outgrowth (diluted, in some cases) were plated onto selective plates and incubated overnight at 37°C. Colonies were counted, adjusted for plating dilution, and graphed.

    Experience Extreme Purity with NEB’s T4 DNA Ligase

    Equivalent amounts of protein were loaded and silver stained using SilverXpress®. Marker M is NEB's Broad Range Protein Marker (NEB #P7702).
    SilverXpress® is a registered trademark of Life Technologies, Inc.