T4 RNA Ligase 2, truncated K227Q |NEB酶试剂 New England Biolabs

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产品信息

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).

产品来源

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.

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
    • M0351S     -20    
        T4 RNA Ligase 2, truncated K227Q M0351SVIAL -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
    • M0351L     -20    
        T4 RNA Ligase 2, truncated K227Q M0351LVIAL -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 (NEB #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

    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

    分子量

    理论上的: 71406.97 daltons

    比活度

    200,000 units/mg

    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

    • 低分子量 ssRNA Ladder
    • microRNA Marker
    • 小鼠 RNase 抑制剂
    • s1315-universal-mirna-cloning-linker

    单独销售的组分

    • T4 RNA 连接酶反应缓冲液

  • 注意事项

    1. 在我们的测试中，此连接酶可以与 T4 Rnl2tr（NEB #M0242）互换使用，但可能需要增加温育时间。添加 PEG 可促进连接（参阅“常见问题解答”）。

  • 参考文献

    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.

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 K227Q (NEB #M0351)

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. 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?
    7. Is PEG 8000 available for purchase?

  • 实验技巧

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