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  • Thermostable 5´ AppDNA/RNA Ligase

    Description

    Thermostable 5´ App DNA/RNA Ligase is a point mutant of catalytic lysine of RNA ligase from Methanobacterium thermoautotrophicum (1). This enzyme is ATP independent. It requires a 5´ pre-adenylated linker for ligation to the 3´-OH end of either RNA or single stranded DNA (ssDNA). The enzyme is also active in ligation of RNA with 2´-O-methylated 3´ end to 5´-adenylated linkers (1). The optimal temperature for ligation reaction is 60–65°C (2). The mutant ligase is unable to adenylate the 5´-phosphate of RNA or ssDNA, which reduces the formation of undesired ligation products (concatemers and circles).

    The ability of the ligase to function at 65°C might reduce the constraints of RNA secondary structure in RNA ligation experiments.

    Product Source

    Thermostable 5´ App DNA/RNA Ligase is expressed as His-tag fusion in E. coli.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 1-2010X
    MnCl210X

    Advantages and Features

    Applications

    • Ligate a 5´ pre-adenylated 3´-blocked DNA or RNA sequence tag to the 3´-OH of RNA and ssDNA.

    Properties and Usage

    Reaction Conditions

    1X NEBuffer 1
    Incubate at 65°C

    1X NEBuffer 1:
    10 mM Bis-Tris-Propane-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    50 mM KCl
    10 mM Tris-HCl
    0.1 mM EDTA
    1 mM DTT
    50% Glycerol
    pH 7.5 @ 25°C

    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.
    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • 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. For optimal ligation of ssDNA to adenylated DNA linkers, we recommend using NEBuffer 1 supplemented with manganese. For ligation of ssRNA to adenylated DNA linkers, just use NEBuffer 1. Heat inactivation or Proteinase K treatment is required to release RNA bound by the ligase.

    References

    1. Zhelkovsky, A. M., McReynolds, L. A. (2012). BMC Mol. Biol.. 13, 24.
    2. Torchia, C., Takagi, Y. and Ho, C. K. (2008). Nucleic Acids Res.. 36, 6218-6227.

    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 can you synthesize an adenylated DNA linker?
    2. Can I use the wild type Mth RNA ligase included in the 5’ DNA adenylation kit instead of the Thermostable 5’AppDNA/RNA ligase for library construction?
    3. How much Thermostable 5’AppDNA/RNA ligase do I need to add to a reaction?
    4. Why do you recommend adding MnCl2 to the DNA ligation but not the RNA reaction?
    5. Does the Thermostable 5’ AppDNA/RNA ligase ligate dsRNA or dsDNA?
    6. After ligation why do you recommend heating at 90°C for 3 minutes or treating with Proteinase K?
    7. Will PEG or increased time enhance the amount of ligated product?
    1. Protocol for ssDNA/RNA Ligation (M0319)

    Selection Tools

    Which ligase should I use?