T4 RNA Ligase 2, truncated |NEB酶试剂 New England Biolabs

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产品信息

T4 RNA Ligase 2, truncated (T4 Rnl2 truncated) specifically ligates the pre-adenylated 5´ end of DNA or RNA to the 3´ end of RNA. The enzyme does not require ATP for ligation but does need the pre-adenylated substrate. T4 Rnl2 truncated is expressed from a plasmid in E. coli which encodes the first 249 amino acids of the full length T4 RNA Ligase 2. Unlike the full length ligase, T4 Rnl2 truncated is unable to adenylate the 5´ end of the substrate, and as a result it cannot ligate the phosphorylated 5´ end of RNA or DNA to the 3´ end of RNA (1-3). This enzyme, also known as Rnl2 (1-249) has been used for optimized linker ligation for the cloning of microRNAs. This enzyme reduces background ligation because it can only use adenylated primers (4-5).

产品来源

An E. coli strain that carries the cloned truncated T4 RNA Ligase 2 gene.

产品类别:
RNA Ligation

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • M0242S     -20    
        T4 RNA Ligase 2, truncated M0242SVIAL -20 1 x 0.01 ml 200,000 units/ml
        T4 RNA Ligase Reaction Buffer B0216SVIAL -20 1 x 1.5 ml 10 X
        PEG 8000 B1004SVIAL -20 1 x 1 ml 1 X
    • M0242L     -20    
        T4 RNA Ligase 2, truncated M0242LVIAL -20 1 x 0.05 ml 200,000 units/ml
        T4 RNA Ligase Reaction Buffer B0216SVIAL -20 1 x 1.5 ml 10 X
        PEG 8000 B1004SVIAL -20 1 x 1 ml 1 X

  • 特性和用法

    单位定义

    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

    反应条件

    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)

    贮存溶液

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

    热失活

    65°C for 20 minutes

    单位活性检测条件

    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.

  • 优势和特性

    应用特性

    • 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

  • 相关产品

    相关产品

    • m0351-t4-rna-ligase-2-truncated-k227q
    • 小鼠 RNase 抑制剂

    单独销售的组分

    • T4 RNA 连接酶反应缓冲液

  • 参考文献

    1. Ho, C.K. et al. (2004). Structure. 12, 327-339.
    2. Ho, C.K. and Shuman, S. (2002). Proc. Natl.Acad.Sci. USA. 99, 12709-12714.
    3. Nandakumar, J. et al. (2004). J. Biol. Chem. 279, 31337-31347.
    4. Aravin, A. and Tusch, T. (2005). FEBS Letters. 579, 5830-5840.
    5. Pfeffer, S. et al. (2005). Nat. Meth.. 2, 269-276.

操作说明、说明书 & 用法

  • 操作说明

    1. Protocol for ligation of the 3´ OH of RNA to the 5´ pre-adenylated DNA with T4 RNA Ligase 2 truncated (NEB #M0242)

工具 & 资源

  • 选择指南

    • Properties of DNA and RNA Ligases
    • RNA Ligase Selection Chart

FAQs & 问题解决指南

  • FAQs

    1. How can I increase ligation efficiency?
    2. Is PEG 8000 available for purchase?

  • 实验技巧

    Since pre-adenylated oligos are used in the reaction, the ligation efficiency is very high. Usually 1-2 hr at 25C is sufficient to achieve desirable results.