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  • T3 DNA Ligase


    T3 DNA Ligase is an ATP-dependent ds DNA ligase from bacteriophage T3. It will catalyze the formation of a phosphodiester bond between adjacent 5´ phosphate and 3´ hydroxyl groups of duplex DNA. Cohesive ends, blunt ends, and nick sealing can all be efficiently catalyzed by T3 DNA Ligase (1). As with T4 DNA Ligase, blunt end ligation is enhanced by the addition of PEG 6000 to the reaction. T3 DNA Ligase exhibits a higher tolerance (2-fold) for NaCl in the reaction compared to T4 DNA Ligase, making the enzyme a versatile choice for in vitro molecular biology protocols requiring DNA ligase activity.

    Product Source

    An E. coli strain containing a recombinant gene encoding T3 DNA Ligase.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    T3 DNA Ligase Reaction Buffer2X

    Advantages and Features


    • Cloning of DNA fragments generated by restriction enzyme digestion
    • Cloning of PCR products
    • Adding linkers or adapters to dsDNA
    • Circularization of linear DNA
    • Nick-sealing in dsDNA
    • Site-directed mutagenesis

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to give 50% ligation of 100 ng HindIII fragments of λ DNA in a total reaction volume of 20 μl in 1 minute at 25°C in 1X T3 DNA Ligase Reaction Buffer.

    Reaction Conditions

    1X T3 DNA Ligase Reaction Buffer
    Incubate at 25°C

    1X T3 DNA Ligase Reaction Buffer:
    66 mM Tris-HCl
    10 mM MgCl2
    1 mM ATP
    1 mM DTT
    7.5% Polyethylene glycol (PEG 6000)
    pH 7.6 @ 25°C

    Storage Temperature


    Storage Conditions

    10 mM Tris-HCl
    50 mM KCl
    1 mM DTT
    50% Glycerol
    0.1 mM EDTA
    pH 7.4 @ 25°C

    Heat Inactivation


    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • RNase Activity (16 Hour Digestion):

      The product is tested in a reaction containing a RNA substrate.  After incubation for 16 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.


    1. ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA Ligase which requires NAD.

      Dilution of enzyme for long-term storage at -20°C should be performed with the storage buffer containing 50% glycerol. Diluent A (NEB #B8001) can also be used for those application in which BSA, present in Diluent A, will not interfere.

      T3 DNA Ligase is also active in buffers without PEG 6000, such as our T4 DNA Ligase Buffer and NEBuffers 1–4, for applications in which PEG 6000 is detrimental. Please remember to supplement the reaction with 1 mM ATP (final concentration). In these buffers T3 DNA Ligase exhibits an approximately 10-fold reduction in activity. In applications where a high concentration of NaCl needs to be maintained, we suggest using a reaction buffer without PEG 6000.

      Heating a reaction containing T3 DNA Ligase at 65°C for 10 minutes will inactivate the enzyme. However, the reaction needs to be performed in a buffer without PEG. Do not heat inactivate if there is PEG in the reaction buffer, as transformation will be inhibited.

    2. Standard vector + insert ligations in 10–20 μl reaction volumes are usually performed for 30 minutes at 25°C.


    1. Cai, L. et al. (2004). J. Biochem. 135, 397-403.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.


    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What type of DNA ends can T3 DNA Ligase ligate?
    2. Can T3 DNA Ligase be used with a buffer that does not contain PEG?
    3. Does T3 DNA Ligase have a higher tolerance to salt than T4 DNA Ligase?
    4. Can T3 DNA Ligase be heat inactivated?
    5. What are some potential problems with the ligation reaction using T3 DNA Ligase that can lead to transformation failure?
    6. What are some other problems that should be considered when trouble shooting a transformation problem?
    7. What problems can be encountered in the restriction digest that can cause ligation using T3 DNA Ligase or subsequent transformation to fail?
    8. What controls should be run to test the cells and DNA when using T3 DNA Ligase?
    9. How much DNA should be used in a ligation using T3 DNA Ligase?
    1. Protocol for T3 DNA Ligase (M0317)

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