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  • T4 RNA Ligase 2, truncated K227Q

    Description

    T4 RNA Ligase 2, truncated K227Q (T4 Rnl2tr K227Q) specifically ligates the pre-adenylated 5´ end of DNA or RNA to the 3´ OH end of RNA. The enzyme does not use ATP for ligation but requires pre-adenylated linkers. 

    T4 Rnl2tr K227Q is a point mutant of T4 RNA Ligase 2, truncated (NEB# M0242) . Mutation of K227 in T4 RNA Ligase 2 reduces enzyme lysyl adenylation (1). This mutation further reduces the formation of undesired ligation products (concatemers and circles) by T4 Rnl2tr (2), possibly by reducing the trace activity of T4 Rnl2tr in transfer of adenylyl groups from linkers to the 5´-phosphates of input RNAs. 

    The exclusion of ATP, use of pre-adenylated linkers, and the reduced enzyme lysyl adenylation activity provide the lowest possible background in ligation reactions. This enzyme has been used for optimized linker ligation for the cloning of microRNAs (2).

    Product Source

    T4 Rnl2tr K227Q is expressed as an MBP fusion from a plasmid in E. coli which encodes the first 249 amino acids of the full length T4 RNA Ligase 2 with a lysine to glutamine mutation at position 227.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    T4 RNA Ligase Reaction Buffer 10X
    PEG 800050%

    Advantages and Features

    Applications

    • Ligate a pre-adenylated DNA or RNA sequence tag to any RNA 3´-end
    • Join a single stranded adenylated primer to small RNAs for cDNA library creation
    • Join a single stranded adenylated primer to RNA for strand-specific cDNA library construction

    Properties and Usage

    Unit Definition

    200 units is defined as the amount of enzyme required to give 80% ligation of a 31-mer RNA to the pre-adenylated end of a 17-mer DNA universal miRNA cloning linker (#S1315) in a total reaction volume of 20 µl in 1 hour at 25°C. 

    5´-FAM- rArGrUrCrGrUrArGrCrCrUrUrUrArUrCrCrGrArGrArUrUrCrArGrCrArArUrA-3´ 

    5´-rAppCTGTAGGCACCATCAAT–NH2-3´ 

    Molarity:
    14 µM

    Concentration:
    200,000 units/ml

    Reaction Conditions

    1X T4 RNA Ligase Reaction Buffer 
    Incubate at 25°C

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

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    65°C for 20 min

    Molecular Weight

    Theoretical: 71406.97 daltons

    Specific Activity

    200,000 units/mg

    Unit Assay Conditions

    1X T4 RNA Ligase Reaction Buffer supplemented to 10% (w/v) PEG MW 8000, 20 pmol of 5´-FAM labeled RNA, and 40 pmol preadenylated DNA linker. After incubation at 25°C for 1 hour, the ligated product is detected on a 15% denaturing polyacrylamide gel.

    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 (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. In our tests, this ligase can be used interchangeably with T4 Rnl2tr (NEB #M0242), but may require increased incubation time. Ligation activity is stimulated by adding PEG (See FAQ).

    References

    1. Yin, S., Ho, C.K. and Shuman S. (2003). J. Biol. Chem. 278, 17601-17608.
    2. Hafner, M., et al. (2008). Methods. 44, 3-12.
    3. Viollet, S., et al. (2011). BMC Biotechnology. 11, 72.

    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. The molecular weight of T4 Rnl2tr K227Q is 71.6 kDa. Is it a fusion?
    2. Can T4 Rnl2tr K227Q be used in other NEBuffers?
    3. Are there differences in ligation efficiency for T4 Rnl2tr and T4 Rnl2tr K227Q?
    4. What can T4 Rnl2tr K227Q ligate?
    5. Does PEG stimulate ligation efficiency?
    6. How can I increase ligation efficiency?
    1. Guidelines for ligation of single-stranded pre-adenlyated DNA adapters to the 3’-ends of single-stranded RNA

    Selection Tools

    Compared to T4Rnl2tr truncated, this mutant produces less side products.