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  • E. coli DNA Ligase

    Description

    Catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA containing cohesive ends.

    Highlights

    • Specific Activity: 6,000 units/mg
    • Supplied with 10X Reaction Buffer

    Product Source

    Purified from E. coli strain 594(su-) carrying the prophage λgt4 lop11 lig+Sam 7 (1) by the procedure of Panasenko et al. (2).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    E. coli DNA Ligase Reaction Buffer-2010X

    Advantages and Features

    Applications

    • Okayama and Berg cDNA cloning (3)

    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 E. coli DNA Ligase Reaction Buffer.

    Reaction Conditions

    1X E. coli DNA Ligase Reaction Buffer
    Incubate at 16°C

    1X E. coli DNA Ligase Reaction Buffer:
    30 mM Tris-HCl
    4 mM MgCl2
    26 μM NAD
    1 mM DTT
    50 μg/ml BSA
    pH 8 @ 25°C

    Usage Concentration

    10,000 units/ml

    Storage Temperature

    -20°C

    Storage Conditions

    300 mM NaCl
    10 mM Tris-HCl
    1 mM DTT
    0.1 mM EDTA
    500 μg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Specific Activity

    6,000 units/mg

    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.
    • 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, DNA):
      The DNA is tested in a reaction under standard reaction conditions. After incubation for 16 hours there is no detectable degradation of the DNA as determined by agarose gel electrophoresis.

    Notes

    1. Requires NAD+ (nicotinamide adenine dinucleotide) as a cofactor, in contrast to other ligases which use rATP.
    2. Ligation of blunt-ended fragments is extremely inefficient. For ligation of blunt-ended fragments use T4 DNA Ligase.
    3. Does not ligate RNA to DNA (4).
    4. This enzyme ligates only DNA fragments with cohesive termini.

    References

    1. Panasenko, S.M. et al. (1977). Science. 196, 188-189.
    2. Panasenko, S.M. et al. (1978). J. Biol. Chem.. 253, 4590-4592.
    3. Okayama, H. and Berg, P. (1982). Mol. Cell. Biol.. 2, 161-170.
    4. Higgins, N.P. and Cozzarelli, N.R. (1979). R. Wu(Ed.), Methods Enzymol.. 68, 50-71. New York: Academic Press.

    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. How does E. coli DNA Ligase (NEB# M0205) differ from T4 DNA Ligase (NEB# M0202)?
    2. Which ligase should I use?
    1. E. coli DNA Ligase Protocol

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    Usage Guidelines & Tips

    Troubleshooting Guides