标签归档：dnase

DNase I 反应缓冲液 |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

NEB 公司为每种酶都提供 10X 反应缓冲液。1X 反应缓冲液浓度确保该酶达到最佳活性。

产品类别:
Exonucleases and Non-specific Endonucleases Products,
Buffers 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
    • B0303S     -20    
        DNase I Reaction Buffer B0303SVIAL -20 4 x 1.5 ml 10 X

  • 特性和用法

    1X 缓冲液组分

    10 mM Tris-HCl
    2.5 mM MgCl2
    0.5 mM CaCl2
    pH 7.6@25°C

  • 相关产品

    相关产品

    • m0303-dnase-i-rnase-free

工具 & 资源

  • Web 工具

    • Exo Selector

FAQs & 问题解决指南

  • FAQs

    1. Will DNase I work in NEB buffers 1-4?
    2. Do you recommend a cleanup step after a DNaseI reaction?
    3. Can I use an RNase inhibitor with my reaction?

DNase I (RNase-free) |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

DNase I, (RNase-free) is an endonuclease that nonspecifically cleaves DNA to release di-, tri- and oligonucleotide products with 5´-phosphorylated and 3´-hydroxylated ends (1,2). DNase I acts on single- and double-stranded DNA, chromatin and RNA:DNA hybrids.

重点

Isolated from a recombinant source
Supplied with 10X Reaction Buffer

产品来源

An E. Coli strain that carries an MBP fusion clone of Bovine Pancreatic DNase I.

产品类别:
Exonucleases and Non-specific Endonucleases Products,
RNA Synthesis In vitro Transcription (IVT)

  • 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
    • M0303S     -20    
        DNase I (RNase-free) M0303SVIAL -20 1 x 0.5 ml 2,000 units/ml
        DNase I Reaction Buffer B0303SVIAL -20 1 x 1.5 ml 10 X
    • M0303L     -20    
        DNase I (RNase-free) M0303LVIAL -20 2 x 1.25 ml 2,000 units/ml
        DNase I Reaction Buffer B0303SVIAL -20 1 x 1.5 ml 10 X

  • 特性和用法

    单位定义

    One unit is defined as the amount of enzyme which will completely degrade 1 µg of pBR322 DNA in 10 minutes at 37°C in DNase I Reaction Buffer.

    Complete degradation is defined as the reduction of the majority of DNA fragments to tetranucleotides or smaller.

    反应条件

    1X DNase I Reaction Buffer
    Incubate at 37°C

    1X DNase I Reaction Buffer
    10 mM Tris-HCl
    2.5 mM MgCl2
    0.5 mM CaCl2
    (pH 7.6 @ 25°C)

    使用浓度

    2,000 units/ml

    贮存溶液

    10 mM Tris-HCl
    2 mM CaCl2
    50% Glycerol
    pH 7.6 @ 25°C

    热失活

    75°C for 10 minutes

  • 优势和特性

    应用特性

    • Degradation of DNA template in transcription reactions
    • Removal of contaminating genomic DNA from RNA samples
    • DNase I footprinting
    • Nick Translation

  • 相关产品

    单独销售的组分

    • DNase I 反应缓冲液

  • 注意事项

    1. EDTA should be added to a final concentration of 5 mM to protect RNA from being degraded during enzyme inactivation (3).
    2. We do not recommend using NEB's DNase I as a substitute for Monarch DNase I in the RNA isolation workflow when using the Monarch Total RNA Miniprep Kit (NEB #T2010).  Monarch DNase I is optimized for use in this workflow, and is available for purchase as a component of the Monarch Total RNA Enzyme Pack, (NEB #T2019).

  • 参考文献

    1. Kunitz, M. (1950). J. Gen. Physiol . 33, 349-362.
    2. Vanecko, S. and laskowski, M. (1961). J. Biol. Chem . 236, 3312-3316.
    3. Huang, Z. et al. (1996). Biotechniques . 20, 1012-1020.

操作说明、说明书 & 用法

  • 操作说明

    1. A Typical DNase I Reaction Protocol (M0303)

  • 使用指南

    • Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

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

FAQs & 问题解决指南

  • FAQs

    1. What is the specific activity of DNase I(RNase-free)?
    2. Will DNase I work in NEB buffers 1-4?
    3. What is the best way to remove DNase I from my reaction?
    4. Will DNase I work in rCutSmart buffer?
    5. Can I use the Monarch RNA Cleanup Kit to cleanup up my DNase I-treated RNA?
    6. Can I use NEB DNase I (#M0303) with the Monarch Total RNA Miniprep Kit?

DNase I-XT |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

DNase I-XT，一种经过基因工程改造的 DNase I 变体，是一种耐盐型 DNA 核酸内切酶，可以非特异性切割 DNA，释放具有 5′ 磷酸化和 3′ 羟基化末端的二核苷酸、三核苷酸和寡核苷酸产物（1, 2）。DNase I-XT 作用于单链和双链 DNA、染色质和 RNA:DNA 杂交体中的 DNA 链。虽然 DNase I（无 RNase)（NEB #M0303）在 > 50 mM 的盐浓度下活性会受到抑制，但 DNase I-XT（NEB #M0570）在 50 – 100 mM 的盐浓度下可表现出最佳活性，并在 200 和 300 mM 的盐浓度下可分别保留 65% 和约 40% 的活性。DNase I-XT 具有更高的耐盐性，使其成为从体外转录（IVT）反应体系中去除 DNA 模板的首选酶。重要的是，DNase I-XT 不含 RNase，可以从 RNA 制备反应中完全去除 DNA，同时保持 RNA 的完整性。

图 1：DNase I-XT 可在 > 100 mM 的盐浓度下有效降解 DNA

DNase I-XT |

对 DNase I（NEB #M0303）和 DNase I-XT（NEB #M0570）的 DNase 活性进行等摩尔比较，与 DNase I 相比，DNase I-XT 展现出更高的耐盐特性。在 1X DNase I 反应缓冲液中，消化标记有荧光和淬灭基团的发夹 dsDNA 底物（35 nt），荧光强度反映出不同盐浓度条件下的酶活（如图所示）。DNase I 活性随着盐浓度的增加而逐步降低，而 DNase I-XT 在盐含量高达 300 mM 的溶液中仍保持活性。

图 2：DNase I-XT（耐盐）能够更完全的去除 IVT 反应和 RNA 制备反应中的 DNA

DNase I-XT |

在以下条件下处理体外转录反应体系（20 μl）：1）无 DNase I；2）2 U TURBO® DNase 或 3）2 U DNase I-XT，在 37℃ 条件下处理 15 分钟。然后使用 Monarch® RNA 纯化试剂盒（500 µg，NEB #T2050）对每个样本进行纯化，并用无核酸酶水（50 μl）中洗脱样本。使用 Luna® 通用探针法 qPCR 预混液（NEB #M3004）通过 qPCR 检测 DNA 残留水平。通过将每个样本的平均 Cq（循环数）值与标准曲线（灰色）进行比较，从而确定残留的可 PCR-扩增 DNA 的百分比。TURBO DNase 和 DNase I-XT 都不需要在 DNase 酶切之前稀释 IVT 反应体系，但是，当使用 DNase I-XT 进行处理时，会从 IVT 反应体系中去除 DNA 模板效果更彻底，不会通过 qPCR 检测到。

图 3：DNase I-XT 可有效去除 RNA 粗提产物中残留的基因组 DNA

DNase I-XT |

对 RNA 粗提样本进行以下任一种处理：1）无 DNase 处理；2）使用溶于 DNase I-XT 反应缓冲液中的 DNase I-XT（2 U，NEB #M0570）；3）使用溶于 DNase I 反应缓冲液中的 DNase I（2 U，NEB #M0303）或 4）使用溶于 TURBO DNase 反应缓冲液中的 TURBO DNase（2 U，ThermoFisher），在 37℃ 条件下处理 15 分钟。使用 Luna 通用一步法 RT-qPCR 试剂盒（NEB #E3005）和人或小鼠 actin 外显子引物，通过 RT-qPCR 对残留基因组 DNA（gDNA）进行定量（+/– RT）。以 qPCR（无反转录，仅扩增DNA）与 RT-qPCR（有反转录，同时扩增 DNA 和 RNA）平均 Cq 值的差值评估 DNase 去除 DNA 的效果。 结果显示：与 DNAse I 或 TURBO DNase 相比，DNase I-XT 从 RNA 粗提产物中去除污染 gDNA 更加彻底。

 

产品来源

A His-tagged engineered variant of DNase I expressed in Pichia pastoris.

产品类别:
Exonucleases and Non-specific Endonucleases Products,
RNA Synthesis In vitro Transcription (IVT)

  • 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
    • M0570S     -20    
        DNase I-XT M0570SVIAL -20 1 x 0.5 ml 2,000 units/ml
        DNase I-XT Reaction Buffer B0570SVIAL -20 1 x 1.5 ml 10 X
    • M0570L     -20    
        DNase I-XT M0570LVIAL -20 2 x 1.25 ml 2,000 units/ml
        DNase I-XT Reaction Buffer B0570SVIAL -20 1 x 1.5 ml 10 X

  • 特性和用法

    单位定义

    1 单位是指在 50 µl 反应体系中，30℃ 条件下，1 分钟内从 35 mer FAM-BHQ1 标记的发夹结构寡核苷酸释放 210 pmol FAM 所需的酶量。

    反应条件

    1X DNase I-XT 反应缓冲液
    Incubate at 37°C

    1X DNase I-XT 反应缓冲液
    10 mM Tris-HCl
    50 mM NaCl
    10 mM MgCl2
    0.5 mM CaCl2
    0.005% Tween® 20
    (pH 7.6 @ 25°C)

    贮存溶液

    10 mM Tris-HCl
    2 mM CaCl2
    50% Glycerol
    pH 7.6 @ 25°C

    热失活

    否

  • 优势和特性

    Features

    • 可在含盐量较高的反应体系中使用，例如 IVT 和 RNA 样本制备
    • 可直接加入到 IVT 反应体系中，无需稀释。
    • 可从 IVT 反应体系和 RNA 样本制备中有效去除 DNA
    • 不含 RNase 的酶，可耐受较宽范围的盐浓度条件（高达 300 mM）

  • 相关产品

    相关产品

    • m0303-dnase-i-rnase-free
    • DNase I 反应缓冲液
    • e2050-hiscribe-t7-quick-high-yield-rna-synthesis-kit
    • T2040 Monarch RNA Cleanup Kit 50 ug

  • 注意事项

    1. DNase I-XT 附带了经过优化的反应缓冲液，用于 DNase I-XT 的反应。为了实现最佳活性，推荐将 DNase I-XT（NEB #M0570）与 DNase I-XT 反应缓冲液（NEB #B0570）搭配使用，而不是使用 DNase I 反应缓冲液（NEB #B0303）。同样，由于 DNase I-XT 反应缓冲液中的盐对 DNase I 会产生抑制作用，不推荐将 DNase I-XT 反应缓冲液（NEB #B0570）与 DNase I（NEB #M0303）搭配使用。
    2. 使用 Monarch 总 RNA 小量提取试剂盒（NEB #T2010）时，不推荐在 RNA 提取实验流程中使用 NEB 的 DNase I-XT 替代 Monarch DNase I。Monarch DNase I 针对此实验流程进行了优化，可作为 Monarch 总 RNA 小量提取酶套装（NEB #T2019）的一个组分售出。

  • 参考文献

    1. Kunitz, M. (1950).  J. Gen. Physiol.. 33, 349-362.
    2. Vanecko, S. and laskowski, M. (1961).  J. Biol. Chem.. 236, 3312-3316.

操作说明、说明书 & 用法

  • 操作说明

    1. Typical DNase I-XT Reaction Protocol (NEB #M0570)
    2. DNA Template Removal from in vitro transcription (IVT) Reaction Using DNase I-XT (NEB #M0570)
    3. Removal of gDNA from RNA Preparations Using DNase I-XT (NEB #M0570)

  • 使用指南

    • Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

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

FAQs & 问题解决指南

  • FAQs

    1. What is the difference between DNase I (RNase-free) (NEB #M0303) and DNase I-XT (NEB #M0570)?
    2. Will DNase I-XT degrade more than 1 µg of DNA in an IVT reaction?
    3. Will DNase I-XT work in NEBuffer r1.1, NEBuffer r2.1, NEBuffer r3.1, or rCutSmart?
    4. What is the best way to remove DNase I-XT from my reaction?
    5. Will DNase I-XT work in rCutSmart buffer?
    6. Can I use the Monarch RNA Cleanup Kit (NEB #T2040) to clean up my DNase I-XT-treated RNA?
    7. Can I use NEB DNase I-XT with the Monarch Total RNA Miniprep Kit (NEB #T2010)?

Duplex DNase |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

Duplex DNase is an engineered double-strand-specific DNA endonuclease that preferentially degrades double-stranded DNA (dsDNA) over single-stranded DNA (ssDNA) or RNA. It will also cleave the DNA strand of a DNA:RNA hybrid duplex.

Figure 1: Duplex DNase preferentially degrades dsDNA over ssDNA

Duplex DNase |

Activity on a dsDNA substrate (35 nt dsDNA hairpin) or ssDNA substrate (15 nt ssDNA) was measured by monitoring the increase in fluorescence when the substrate is degraded by Duplex DNase. FLUOR = Fluorophore; QUENCH = Quencher

Figure 2: Duplex DNase is more specific for dsDNA than DNase I

Duplex DNase |

Activity of Duplex DNase (NEB #M7635) or DNase I (NEB #M0303) on a dsDNA substrate (35 nt dsDNA hairpin) or ssDNA substrate (15 nt ssDNA) was measured by monitoring the increase in fluorescence when the substrate is degraded by either enzyme in their respective reaction buffers at 30°C. Duplex DNase is > 5-fold more specific than DNase I is for dsDNA. The ratio of activity on double-stranded vs. single-stranded substrates demonstrates the increased specificity that Duplex DNase shows for dsDNA over ssDNA as compared to DNase I.

Figure 3: Duplex DNase degrades the DNA strand of a DNA:RNA hybrid

Duplex DNase |

An in vitro transcription reaction (50 μl) was treated with 2 U DNase I-XT for 15 minutes at 37°C to digest template DNA. The RNA was purified using the Monarch® RNA Cleanup Kit (500 µg, NEB #T2050) and eluted in nuclease-free water (50 μl). Varying amounts (from 106-1012 copies) of Cluc template RNA were mixed with a 5´ fluorescently-labeled/3´-quenched 22 nt complementary DNA probe (0.2 µM final concentration, Tm 61.8°C) in NEBuffer™ r1.1. Duplex DNase (2U) was added, the reaction incubated at 58°C, and the relative fluorescence was monitored every 10 seconds in a Bio-Rad® CFX Touch™ qPCR instrument. The increase in fluorescence observed over time (which is not observed in the non-template control) demonstrates the ability of Duplex DNase to cleave the fluorescently labeled DNA strand of a DNA:RNA hybrid (probe:Cluc RNA). Moreover, the cleavage of the DNA probe when hybridized to the template RNA increases over time and is dose-dependent on the amount of Cluc template RNA present. FLUOR = Fluorophore; QUENCH = Quencher

Figure 4: Duplex DNase preferentially degrades dsDNA in a mixture of dsDNA and ssDNA over a wide range of temperatures

Duplex DNase |

A mixture of 20 pmol of dsDNA (60 bp oligonucleotide) and 20 pmol of ssDNA (50 nt oligonucleotide) was incubated with 2 Units of Duplex DNase for 1 minute at 25°C, 37°C, 50°C or 65°C and then run on a 4% E-gel™ EX gel (Thermo Fisher Scientific®) stained with SYBR® Gold. Lane 1: dsDNA PCR Marker (NEB #N3234), Lane 2: ssDNA 50 nt, Lane 3: dsDNA 60 bp, Lanes 4–7: 25°C, 37°C, 50°C, 65°C. Duplex DNase displays increased activity and preference for double stranded DNA with increasing temperatures. FLUOR = Fluorophore

产品来源

A His-tagged engineered Duplex DNase expressed in Pichia pastoris.

产品类别:
Exonucleases and Non-specific Endonucleases Products

应用:
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
    • M7635S     -20    
        Duplex DNase M7635SVIAL -20 1 x 0.075 ml 2,000 units/ml
        NEBuffer™ r1.1 B6001SVIAL -20 1 x 1.25 ml 10 X
    • M7635L     -20    
        Duplex DNase M7635LVIAL -20 1 x 0.375 ml 2,000 units/ml
        NEBuffer™ r1.1 B6001SVIAL -20 2 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 release 50 pmol of FAM from a 35mer FAM-BHQ1 labeled hairpin oligonucleotide in 1 minute at 30°C in a 50 µl reaction volume.

    反应条件

    1X NEBuffer™ r1.1

    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)

    使用浓度

    2,000 units/ml

    贮存溶液

    10 mM Tris-HCl
    50 mM KCl
    50% Glycerol
    pH 7.4 @ 25°C

    热失活

    75°C for 10 minutes in the presence of 1 mM DTT. If the sample contains RNA, we recommend adding EDTA (10 mM final concentration) and DTT (1 mM final concentration), prior to heat inactivation. RNA may degrade at temperatures >65°C in the presence of divalent metals such as Mg2+.

  • 相关产品

    相关产品

    • m0303-dnase-i-rnase-free
    • M0570 DNase I-XT

操作说明、说明书 & 用法

  • 操作说明

    1. A Typical Duplex DNase Reaction Protocol (NEB #M7635)

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

FAQs & 问题解决指南

  • FAQs

    1. Can Duplex DNase be heat-inactivated?
    2. Is Duplex DNase inhibited by salt?
    3. At what temperature should I use Duplex DNase?
    4. What is the minimal length of duplex DNA required for Duplex DNase to cleave?
    5.  Does Duplex DNase require divalent cations?
    6. What are the optimal reaction conditions for Duplex DNase?
    7. How does the unit definition for Duplex DNase compare to other duplex-specific DNases?