标签归档：rnase

RNase Inhibitor, Human Placenta |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

RNase Inhibitor, Human Placenta is a recombinant human placental protein which specifically inhibits ribonucleases (RNases) A, B and C (1). It is not effective against RNase 1, RNase T1, S1 Nuclease, RNase H or RNase from Aspergillus. In addition, no inhibition of polymerase activity is observed when RNase Inhibitor, Human Placenta is used with Taq DNA Polymerase, AMV or M-MuLV Reverse Transcriptases, or Phage RNA Polymerases (SP6, T7, or T3).

The 50 kDa protein inhibits RNases by binding noncovalently in a 1:1 ratio with an association constant greater than 1014 (2).

产品来源

An E. coli strain that carries the Ribonuclease Inhibitor gene from human placenta.

产品类别:
RNase Inhibitors,
RNA Synthesis In vitro Transcription (IVT)

应用:
RT-PCR & cDNA Synthesis

  • 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
    • M0307S     -20    
        RNase Inhibitor, Human Placenta M0307SVIAL -20 1 x 0.05 ml 40,000 units/ml
    • M0307L     -20    
        RNase Inhibitor, Human Placenta M0307LVIAL -20 1 x 0.25 ml 40,000 units/ml

  • 特性和用法

    单位定义

    One unit is defined as the amount of RNase Inhibitor, Human Placenta required to inhibit the activity of 5 ng of RNase A by 50%. Activity is measured by the inhibition of hydrolysis of cytidine 2, 3′-cyclic monophosphate by RNase A.

    贮存溶液

    50 mM KCl
    8 mM DTT
    50% Glycerol
    20 mM HEPES-KOH
    pH 7.6 @ 25°C

  • 注意事项

    1. Since ribonucleases typically retain activity under denaturing conditions, care must be taken to avoid denaturing RNase Inhibitor molecules which have complexed with a ribonuclease. To prevent the release of active ribonuclease, temperatures greater than 50°C and high concentrations of urea or other denaturing agents should be avoided.
    2. The recommended concentration of RNase Inhibitor in a reaction is 1 unit/μl. During assembly of a reaction, RNase Inhibitor should be added before other components that are possible sources of RNase contamination (i.e. enzymes, plasmid from a mini prep.)

  • 参考文献

    1. Blackburn, P. and Moore, S. (1982). Pancreatic Ribonucleases . The Enzymes. XV, Part B, Academic Press, NY.
    2. Blackburn, P., Wilson, G. and Moore, S. (1977). J. Biol. Chem. 252,5904.

操作说明、说明书 & 用法

  • 操作说明

    1. Protocol for Avoiding Rnase Contamination using Rnase Inhibitor, Human Placenta (M0307)

Monarch® RNase A |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

Monarch RNase A is a component of the Monarch Genomic DNA Purification Kit (NEB #T3010), which can be used to purify genomic DNA from a variety of sample types. Monarch RNase A degrades single-stranded RNA at C and U residues. This formulation is purified from cow pancreas, is free of contaminating protease or DNase activity, and contains no residual host DNA. It is used at 56°C in the Monarch Genomic DNA Purification Kit protocol but is active at any temperature between 20°C and 65°C.  It is supplied as a 20 mg/ml solution in 50% glycerol, 50 mM Tris-Cl pH 7.4.

Treatment with RNase A is an optional step in the Monarch Genomic DNA Purification Kit protocol for removal of  any residual RNA. Co-purification of RNA during gDNA extractions is a common problem that leads to the overestimation of DNA yield. The low alcohol binding conditions employed in the kit optimizes binding of gDNA alone, so RNA co-purification is extremely low; however, an RNase A treatment step is included in each protocol as an option for the user. 

产品类别:
RNases,
Genomic DNA Extraction & Purification Products,
Nucleic Acid Purification Products

应用:
Nucleic Acid Purification

  • 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
    • T3018L     -20    
        Monarch® RNase A T3018-2 -20 2 x 0.5 ml 20 mg/ml

  • 特性和用法

    贮存溶液

    50 mM Tris-HCl
    50% Glycerol
    pH 7.4

操作说明、说明书 & 用法

  • 操作说明

    1. Genomic DNA Purification from Buccal Swabs (NEB #T3010)
    2. Protocol for Extraction and Purification of Genomic DNA from Blood (NEB #T3010)
    3. Protocol for Extraction and Purification of Genomic DNA from Tissues (NEB #T3010)
    4. Genomic DNA Purification from Yeast (NEB #T3010)
    5. Genomic DNA Purification from Saliva (NEB #T3010)
    6. Protocol for Genomic DNA Cleanup (NEB #T3010)
    7. Genomic DNA Purification from Gram-negative Bacteria (NEB #T3010)
    8. Genomic DNA Purification from Gram-positive Bacteria and Archaea (NEB #T3010)
    9. Protocol for Extraction and Purification of Genomic DNA from Cells (NEB #T3010)
    10. Quick Protocol for Extraction and Purification of Genomic DNA

  • 使用指南

    • Choosing Input Amounts for the Monarch Genomic DNA Purification Kit
    • Factors Affecting DNA Quality when Purifying gDNA from Blood and Tissues with the Monarch Genomic DNA Purification Kit
    • Guidelines for Handling Tissue Samples when using the Monarch Genomic DNA Purification Kit

FAQs & 问题解决指南

  • FAQs

    1. Can I use a different RNase A with the Monarch® Genomic DNA Purification Kit?
    2. Can I omit the RNase digestion step from the protocol? 
    3. Can I use Monarch® RNase A for applications/workflows other than gDNA purification?
    4. The enzymes in the kit were not stored at -20°C right away. Will they still work?

  • 问题解决指南

    • Troubleshooting Guide for Genomic DNA Extraction & Purification (NEB #T3010)

​RNase 4 |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

RNase 4 is a single-stranded RNA endonuclease that cleaves 3´ of uridine in uridine-purine sequences (cut sites: U/A and U/G). RNase 4 allows more targeted digestion of substrate RNA compared to single-nucleotide specific RNases like T1 (cut site: after G), RNase U2 (cut sites: after A and G), or bovine pancreatic RNase A (cut sites: after C and U). RNase 4 endonuclease activity tolerates uridine base modifications such as pseudo-, N1-methyl-pseudo-, dihydro-, and 5-methoxy-uridine species (Ψ, m1Ψ, D, and mo5U). Due to the chemical mechanism of RNase 4 endonucleolytic cleavage, product oligonucleotides contain heterogenous 3´ ends, where most species contain a linear 3´-phosphate or cyclic 2´,3´-phosphate.

Figure 1: RNase 4 cuts at U/A and U/G

​RNase 4 |

Figure 2: RNase 4 enables comprehensive mRNA analysis by LC–MS/MS

​RNase 4 |

Cap: Relative populations of 5´ mRNA ends are identified and measured with the aid of a complementary DNA probe that blocks cleavage by RNase 4. Isolation of the cleaved product prior to LC–MS/MS analysis can be performed using a probe that contains an affinity tag. RNase 4 enables predictable, site-specific generation of 5´ end products with a simple experimental design.
Sequence: To verify the mRNA sequence and its modification status, samples are fully digested with RNase 4 to produce a series of defined oligonucleotides in sizes that are amenable to LC–MS/MS analysis. A coverage map can be generated by aligning the observed oligonucleotides to the reference sequence. One or more additional RNases (e.g., RNase T1) can be used in parallel digestions to increase the total coverage of mRNA.
Tail: RNase 4 can be used to assess the mRNA 3´ end by cutting and releasing the poly(A) tail for length profiling.

Figure 3: RNase 4 improves mRNA sequence characterization by LC–MS/MS

​RNase 4 |

A: Schematic of the digestion workflow with RNase 4. The RNA sample is first heat denatured at 90°C in the presence of 3 M urea (user supplied) for 10 minutes. After dilution to 1 M urea, RNase 4 reactions are performed in 1X NEBuffer™ r1.1 (NEB #M1284), provided as a 10X stock solution. The resulting oligonucleotide pool is directly analyzed by LC–MS/MS.
B: Representative sequence coverage map of FLuc mRNA from two independent experiments with RNase 4 or RNase T1. HiScribe®  T7 (NEB #E2040) was used to generate FLuc mRNA. Colored bars in the coverage map indicate oligonucleotides uniquely assigned to indicated positions along the reference sequence (x-axis). The percentage sequence coverage is reported.

Figure 4: Overview of the mRNA 5´ end analysis workflow with RNase 4

​RNase 4 |

1: A user designed biotinylated DNA oligomer is hybridized to the 5´ end of a complementary RNA sequence upstream of a chosen UA or UG cut site. As RNase 4 is specific for single-strand RNA, any potential cut site within the hybridized DNA:RNA duplex region is protected from cleavage.
2: After RNase 4 digestion, probe hybridized mRNA 5´ end products can be captured via Streptavidin Magnetic Beads (NEB #S1420). 3: Enriched DNA:RNA duplexes are eluted and analyzed by UHPLC–MS/MS

Figure 5: RNase 4 simplifies experimental design and data processing in mRNA 5´-end analysis

​RNase 4 |

A: RNase 4 allows for greater specificity and flexibility in cut site selection compared to RNase H, whose cut site specificity is less predictable. Moreover, RNase H workflows require careful design and optimization of a chimeric RNA-DNA probe, where RNase 4 workflows use a DNA probe. Biotin or desthiobiotin conjugation at the 5´ or 3´ end provides the ability to enrich probe hybridized fragments by streptavidin prior to LC–MS/MS analysis.
B: Representative heatmap of total FLuc mRNA 5´ end cleavage products obtained using RNase 4 or RNase H as outlined in (A) from two independent experiments.
C: Estimated distribution of 5´ end-capped products and intermediates. Error bars represent the standard deviation from two independent experiments.

Figure 6: Reaction conditions for optimal RNase 4 mapping performance can be tuned based on RNA sequence and modification status

​RNase 4 |

Results are plotted for RNAs of 878 (red) or 4,938 nucleotides (blue) in length. A fully m1φ substituted 4,938 nucleotide transcript (yellow) was generated by replacing UTP with cognate m1φTP during in vitro transcription with the HiScribe®  T7 High Yield RNA Synthesis Kit (NEB #E2040). Similar to other RNases used for LC–MS/MS sequence mapping, the amount of RNase 4 to include for maximal sequence coverage should be empirically determined. Generally, longer and more modified RNA requires more units of enzyme for optimal mapping coverage. However, using a large excess of RNase 4, or longer incubation times does not necessarily provide the highest sequence coverage and may result in spurious digestion. RNase 4 (NEB #M1284) is provided as a 50 U/µl solution.

产品来源

An Escherichia coli strain that carries the cloned RNASE4 gene from Homo sapiens, with an N-terminal 6xHis tag.

产品类别:
RNases

  • 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
    • M1284S     -20    
        RNase 4 M1284SVIAL -20 1 x 0.05 ml 50,000 units/ml
        NEBuffer™ r1.1 B6001SVIAL -20 1 x 1.25 ml 10 X
    • M1284L     -20    
        RNase 4 M1284LVIAL -20 1 x 0.25 ml 50,000 units/ml
        NEBuffer™ r1.1 B6001SVIAL -20 1 x 1.25 ml 10 X

  • 特性和用法

    反应条件

    1X NEBuffer™ r1.1
    Incubate at 37°C

    1X NEBuffer™ r1.1
    10 mM Bis-Tris-Propane-HCl
    10 mM MgCl2
    100 µg/ml 重组白蛋白
    (pH 7 @ 25°C)

    贮存溶液

    100 mM NaCl
    50 mM NaOAc
    200 µg/ml Recombinant Albumin
    50% Glycerol
    pH 6 @ 25°C

    热失活

    否

  • 参考文献

    1. Wolf et al. (2022). Human RNase 4 improves mRNA sequence characterization by LC-MS/MS. NAR. PubMedID: 35871301
    2. Wolf et al. (2023). Selective Characterization of mRNA 5′ End-Capping by DNA Probe-Directed Enrichment with Site-Specific Endoribonucleases. ACS Pharmacol. Transl. Sci. PubMedID: 37974627

操作说明、说明书 & 用法

  • 操作说明

    1. RNase 4 (NEB #M1284) DNA Probe-Directed Analysis of mRNA 5´Cap Structures
    2. RNase 4 (NEB #M1284) Digestion Protocol

FAQs & 问题解决指南

  • FAQs

    1. Does RNase 4 (NEB #M1284) cut RNA at sites other than U/A and U/G?
    2. How much RNase 4 (NEB #M1284) should I use to digest RNA for LC-MS/MS nucleotide sequence mapping?
    3. What is the chemical identity of 5´ and 3´ oligonucleotides generated after RNase 4 (NEB #M1284) cleavage?
    4. How much RNase 4 (NEB #M1284) should I use for DNA-probe directed 5´-cap mRNA analysis?
    5. Can RNase 4 (NEB #M1284) be inactivated?
    6. Is RNase 4 (NEB #M1284) endonucleolytic activity sensitive to RNA modifications?
    7. Does RNA secondary structure impact RNase 4 (NEB #M1284) activity?
    8. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

RNase H |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

RNase H（核糖核酸酶 H）是一种核糖核酸内切酶，它能够特异性地水解杂交到 DNA 链上的 RNA 磷酸二酯键。该酶不能消化单链或双链 DNA。

产品来源

大肠杆菌菌株，携带有克隆自大肠杆菌（Escherichia coli）的 RNase H (rnh) 基因。

产品类别:
RNases,
cDNA Synthesis & Reverse Transcriptases Products

应用:
RT-PCR & cDNA Synthesis,
PCR

  • 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
    • M0297S     -20    
        RNase H M0297SVIAL -20 1 x 0.05 ml 5,000 units/ml
        RNase H Reaction Buffer B0297SVIAL -20 1 x 1.5 ml 10 X
    • M0297L     -20    
        RNase H M0297LVIAL -20 1 x 0.25 ml 5,000 units/ml
        RNase H Reaction Buffer B0297SVIAL -20 1 x 1.5 ml 10 X

  • 特性和用法

    单位定义

    1 单位指 50 µl 反应体系中，37℃ 条件下，20 分钟从 20 pmol 荧光标记的 50 bp RNA-DNA 杂交链中水解生成 1 nmol 核苷酸所需的酶量。

    反应条件

    1X RNase H 反应缓冲液
    Incubate at 37°C

    1X RNase H 反应缓冲液
    50 mM Tris-HCl
    75 mM KCl
    3 mM MgCl2
    10 mM DTT
    (pH 8.3 @ 25°C)

    贮存溶液

    50 mM KCl
    10 mM Tris-HCl
    0.1 mM EDTA
    1 mM DTT
    200 µg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    热失活

    65°C for 20 minutes

  • 相关产品

    单独销售的组分

    • RNase H Reaction Buffer

  • 参考文献

    1. Davis, R. et al. (1988). Cell Biol. 8, 4745-4755.
    2. Gubbler, U. and Hoffman, B.J. (1983). Gene. 25, 263-269.
    3. Donnis-Keller, H. (1979). Nucl. Acids Res. 7, 179.
    4. Goodwin, E.C. and Rottman, F.M. (1992). Nucl. Acids Res. 20, 916.

操作说明、说明书 & 用法

  • 操作说明

    1. Cleavage of an RNA strand in an RNA-DNA hybrid with RNase H (NEB #M0297)

FAQs & 问题解决指南

  • FAQs

    1. When I thaw the 10X RNase H Reaction Buffer, I see a white precipitate.  What is it and what should I do about it?
    2. Is DNA Polymerase I active in RNase H buffer?
    3. Which side of the RNA base does RNase H cut?
    4. What is the activity of RNase H at 30°C?