标签归档：truncated

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

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上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

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.

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

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

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.

Exonuclease VIII, truncated |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

产品来源

An E. coli strain that carries a plasmid with genetic engineering active domain of Exonuclease VIII.

产品类别:
Exonucleases and Non-specific Endonucleases Products

  • 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
    • M0545S     -20    
        Exonuclease VIII, truncated M0545SVIAL -20 1 x 0.1 ml 10,000 units/ml
        NEBuffer™ 4 B7004SVIAL -20 1 x 1.25 ml 10 X

  • 特性和用法

    单位定义

    One unit is defined as the amount of enzyme required to produce 1 nmol of acid-soluble deoxyribonucleotide from double-stranded DNA in a total reaction volume of 50 µl in 30 minutes at 37°C in 1X NEBuffer 4 with 0.15 mM sonicated duplex [3H] DNA.

    反应条件

    1X NEBuffer™ 4
    Incubate at 37°C

    1X NEBuffer™ 4
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    1 mM DTT
    (pH 7.9 @ 25°C)

    贮存溶液

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

    热失活

    70°C for 15 minutes

    单位活性检测条件

    1X NEBuffer 4 with 0.15 mM with sonicated duplex 3H- DNA.

  • 优势和特性

    应用特性

    •    Degradation of linear dsDNA while maintaining double stranded circular DNA.

  • 参考文献

    1. Chang, et al. (2001). J. Biol. Chem.. 46004–46010.

操作说明、说明书 & 用法

  • 操作说明

    1. Protocol for Exonuclease VIII, truncated (M0545)

工具 & 资源

  • 选择指南

    • Activities of Exonucleases and Non-specific Endonucleases
    • Common Applications for Exonucleases and Endonucleases
    • Properties of Exonucleases and Non-specific Endonucleases

  • Web 工具

    • Exo Selector

  • 研究摘要

    • Choosing the Right Exonuclease (2019)

FAQs & 问题解决指南

  • FAQs

    1. How does Exonuclease VIII, truncated differ from Lambda Exonuclease (NEB# M0262)?

    2. Will Exonuclease VIII, truncated cleave supercoiled dsDNA or covalently close circular form dsDNA?

    3. How does Exonuclease VIII, truncated differ from Exonuclease III (NEB# M0206)?
    4. Can DNA be blunted using Exonuclease VIII, truncated?
    5. What is the activity of Exonuclease VIII, truncated in these common buffers?