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  • T7 DNA Ligase

    Description

    T7 DNA Ligase is an ATP-dependent ds DNA ligase from bacteriophage T7. It will catalyze the formation of a phosphodiester bond between adjacent 5´ phosphate and 3´ hydroxyl groups of duplex DNA. Cohesive end ligation and nick sealing can be efficiently catalyzed by T7 DNA Ligase (1,2). However, unlike T4 and T3 DNA Ligases, blunt end ligation is not efficiently catalyzed by T7 DNA Ligase. Addition of high concentrations of PEG 6000 [≥ 20% (w/v)] to the reaction can force T7 DNA Ligase to have measurable activity. However, under typical reaction conditions blunt-end DNA ligation does not occur in the presence of T7 DNA Ligase, making it a good choice for applications in which blunt and cohesive ends of DNA are present but only the cohesive ends are to be joined.

    M0318
    T7 DNA Ligase does not ligate blunt-end DNA fragments. Ligation reactions containing blunt fragments (ΦX174 DNA-HaeIII Digest (NEB #N3026 ) and sticky-end fragments (λ DNA-HindIII Digest (NEB #N3012 ) 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.

    Product Source

    An E. coli strain containing a recombinant gene encoding T7 DNA Ligase.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    T7 DNA Ligase Reaction Buffer2X

    Advantages and Features

    Applications

    • Cloning of DNA fragments generated by restriction enzyme digestion
    • 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 30 minutes at 25°C in 1X T7 DNA Ligase Reaction Buffer.

    Reaction Conditions

    1X T7 DNA Ligase Reaction Buffer
    Incubate at 25°C

    1X T7 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

    -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

    No

    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.

    Notes

    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 applications in which BSA, present in Diluent A, will not interfere.

      T7 DNA Ligase is also active in buffers without PEG 6000, such as our T4 DNA Ligase Buffer and NEBuffer 1–4, for applications in which PEG 6000 is detrimental. Please remember to supplement the reaction with 1 mM ATP (final concentration). Using these buffers, the activity of T7 DNA Ligase is reduced approximately 10-fold.

      Heating a reaction containing T7 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.

    References

    1. Doherty, A.J. et al. (1996). J. Biol. Chem. 271, 11083-11089.
    2. Subramanya, H.S. et al. (1996). Cell. 85, 607-615.

    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. T7 DNA Ligase is described as an enzyme that only ligates DNA fragments with cohesive ends. What length of cohesive end can T7 DNA Ligase ligate?
    2. Can T7 DNA Ligase be used with a buffer that does not contain PEG?
    3. Can T7 DNA Ligase be heat inactivated?
    4. What are some potential problems with the ligation reaction using T7 DNA Ligase that can lead to transformation failure?
    5. What are some other problems that should be considered when trouble shooting a transformation problem?
    6. What problems can be encountered in the restriction digest that can cause ligation using T7 DNA Ligase or subsequent transformation to fail?
    7. How much DNA should be used in a ligation using T7 DNA Ligase?
    1. Protocol for T7 DNA Ligase (M0318)

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