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  • T4 DNA Ligase

    Description

      
    Catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1).

    Highlights

    • Isolated from a recombinant source
    • Supplied with 10X Reaction Buffer

    Product Source

    Purified from E. coli C600 pcl857 pPLc28 lig8 (2).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    T4 DNA Ligase Reaction Buffer-2010X

    Advantages and Features

    Applications

    • Cloning of restriction fragments
    • Joining linkers and adapters to blunt-ended DNA

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer.

    Concentration:
    400,000 cohesive end units/ml and 2,000,000 cohesive end units/ml

    Reaction Conditions

    1X T4 DNA Ligase Reaction Buffer
    Incubate at 16°C

    1X T4 DNA Ligase Reaction Buffer:
    50 mM Tris-HCl
    10 mM MgCl2
    1 mM ATP
    10 mM DTT
    pH 7.5 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    65°C for 10 min

    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.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • 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.

    Notes

    1. ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
    2. To dilute T4 DNA Ligase that will subsequently be stored at -20°C, 50% glycerol storage buffer (Diluent Buffer A,NEB #B8001S) should be used; to dilute for immediate use, 1X T4 DNA Ligase Reaction Buffer can be used.
    3. Ligation can also be performed in any of the four restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer if they are supplemented with 1 mM ATP.
    4. Room Temperature Ligation:
      For convenience, ligations may be done at room temperature (20-25°C). For cohesive (sticky) ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 10 minutes. For blunt ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 2 hours or 1 µl high concentration T4 DNA Ligase for 10 minutes. Alternatively, NEB's Quick Ligation Kit (#M2200S) [30 reactions] or (#M2200L) [150 reactions]) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature.

    References

    1. Engler, M.J. and Richardson, C.C. (1982). P.D. Boyer(Ed.), 5, 3. San Diego: Academic Press.
    2. Remaut, E., Tsao, H. and Fiers, W. (1983). Gene. 22, 103-113.
    3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd Ed.). 1.53-1.73.

    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.

    Datacards

    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 are some potential problems with the ligation reaction using T4 DNA Ligase that can lead to transformation failure?
    2. What are some other problems that should be considered when troubleshooting a transformation problem?
    3. What problems can be encountered in the restriction digest that can cause ligation using T4 DNA Ligase or subsequent transformation to fail?
    4. What controls should be run to test the cells and DNA when using T4 DNA Ligase?
    5. When should T4 DNA Ligase be the enzyme of choice?
    6. Can the T4 DNA Ligase be used with the Quick Ligase buffer?
    7. What is the definition of a Weiss Unit and a Cohesive End Unit?
    8. What is the difference between the two definitions and why does NEB use the Cohesive End Unit?
    9. How much DNA should be used in a ligation using T4 DNA Ligase?
    10. Can T4 DNA Ligase be used in other NEBuffers?
    11. Can T4 DNA Ligase be heat inactivated?
    1. Ligation Protocol with T4 DNA Ligase (M0202)

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides