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当前位置: 首页 > 产品中心 > > NEB/WarmStart® Nt.BstNBI/1,000 units/R0725S
商品详细NEB/WarmStart® Nt.BstNBI/1,000 units/R0725S
NEB/WarmStart® Nt.BstNBI/1,000 units/R0725S
NEB/WarmStart® Nt.BstNBI/1,000 units/R0725S
商品编号: R0725S
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产地: 美国(厂家直采)
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产品分类: 其他
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商品介绍

WarmStart Nt.BstNBI is a site specific nicking endonuclease cloned from Bacillus Stereothermophilus. It is formulated with a reversibly-bound aptamer, which inhibits its nicking activity at temperatures below 40°C. WarmStart Nt.BstNBI cleaves only one strand of DNA on a double-stranded DNA substrate. The nicking endonuclease catalyzes a single strand break 4 bases beyond the 3′ side of the recognition sequence.Highlights

  • Nt.BstNBI catalyzes a single-stranded nick 3′ of its recognition sequence. WarmStart functionality is achieved by formulation with a DNA aptamer that inhibits enzyme activity below 40°C
Figure 1: No detectable activity of WarmStart Nt.BstNBI at 25 °C
A) Nicking activity on phage T7 DNA. 1 μg T7 phage DNA was incubated with various amounts of WarmStart Nt.BstNBI (NEB #R0725) and Nt.BstNBI (Non-WarmStart, NEB #R0607) in a 50 μl reaction in NEBuffer r3.1 for 1 hour at 25°C or 55°C. 8 units of enzyme was added and a 2-fold serial dilution was performed. As indicated with “+”, full activity is observed for both enzymes at the permissive temp (55°C) but not at the restrictive temp (25 °C) for the WarmStart version. B) Nicking activity on a fluorescently labeled DNA duplex. 2 pmol 48-bp DNA duplex containing an internal recognition site for Nt.BstNBI was incubated with various amounts of WarmStart Nt.BstNBI (NEB #R0725) and Nt.BstNBI (Non-WarmStart, NEB #R0607) in a 20 μl reaction with NEBuffer r3.1 for 30 min at 25°C or 55°C. 10 units of enzyme was added and a 2-fold serial dilution was performed. The reactions were quenched in 20 mM EDTA, 0.1% Tween 20 and then diluted with water to the final concentration of FAM-labeled DNA duplex at 1 nM. The reactions were analyzed by capillary electrophoresis (CE) fragment analysis on an Applied Biosystems 3730xl Genetic Analyzer (96 capillary array). % Product was determined as the area of the product peak divided by the total area of all peaks in the FAM channel. The average and the standard deviation of % product (Y-axis) plotted with log phase of nicking enzyme units (X-axis) was taken from triplicate reactions. Conclusion: The WarmStart version exhibits similar activity to the regular version at 55°C but has no detectable activity at 25°C.
Figure 2: Reaction temperature profile of WarmStart Nt.BstNBI
Nicking activity of WarmStart Nt.BstNBI (NEB #R0725) and Nt.BstNBI (Non-WarmStart NEB #R0607) on a fluorescently-labeled DNA duplex was measured at various temperatures (25°C to 80°C, 5°C intervals). Reactions containing 0.125 unit WarmStart or non- WarmStart Nt.BstNBI, 100 nM DNA duplex (5’ 6-FAM labeled) in 20 μl NEBuffer r3.1 were incubated for 30 min at various temperatures. The average and the standard deviation of % product (Y-axis) plotted with temperatures (X-axis) was taken from triplicate reactions. Conclusion: WarmStart Nt.BstNBI shows less than 10% activity below 40°C and is undetectable below 30°C, thereby enabling reaction setup at room temperature with no unintended conversion of substrate.
Figure 3: WarmStart Nt.BstNBI facilitates SDA by allowing room temperature setup
Identical Strand Displacement Amplification (SDA) reactions were run either after incubation at 25°C for 30 minutes (to mimic room temperature reaction setup) or immediately after setup on ice, using WarmStart Nt.BstNBI (NEB #R0725) or Nt.BstNBI (Non-WarmStart, NEB #R0607). Reactions were performed in 25 μl of 1X Isothermal Amplification Buffer (NEB #B0537) with 0.5 μl LAMP Fluorescent Dye (NEB #B1700), 0.4 mM dNTPs (NEB #N0447), 10 ng of template DNA (HeLa genomic DNA, 290 copies), 0.5 mM of each primer, 1 μl Bst 2.0 Polymerase (NEB #M0537S) and 1 μl WarmStart Nt.BstNBI or Nt.BstNBI. Isothermal amplification was performed at 55°C and fluorescence was monitored for 45 minutes. The time differential for the appearance of positive signal in test samples and negative controls is distinguishable only for reactions set up on ice (both versions) or at room temperature (WarmStart version only). The variation between technical replicates was low.Conclusion: Only WarmStart Nt.BstNBI enabled successful reaction setup at room temp for SDA reactions targeting a hBRCA1 amplicon in genomic DNA.

Product Source

An E. coli strain that carries the cloned Nt.BstNBI gene from Bacillus stereothermophilus 33M (Z. Chen).
This product is related to the following categories:
Nicking Endonucleases Products,
Restriction Endonucleases: N-O,
Restriction Endonucleases Products
This product can be used in the following applications:
Strand Displacement Amplification & Nicking Enzyme
品牌介绍
New England Biolabs(NEB)公司       NEB公司——三十多年的卓越品质NEB公司成立于二十世纪七十年代中期,拥有众多经验丰富的科学家,是生产生命科学试剂的领导者。目前,NEB为基因组研究提供最齐全的重组酶和天然酶,并且公司业务范围已延伸至蛋白质组学和药品开发领域。回顾三十余年来的历程,NEB公司作为先驱公司之一,为促进生物科技工业的发展做出了巨大的贡献。NEB美国总部乔迁新址后拥有最尖端的设备,有一座现代化的发酵中心及设备齐全的实验室,这些实验室主要用于产品生产、质量监控、产品开发和基础科研之用。作为首批以商业规模生产限制性内切酶的公司之一,NEB一直专注于内切酶的研究,并保持业内领先水平。NEB公司一贯坚持以科学为本的原则,公司生产的试剂因其高质量、高性价比享誉世界。重组酶NEB公司对酶的生产与其基础科研不能分开,通过对限制/修饰系统的克隆和过量表达方面的研究,使我们能够大大降低成本,改善产品质量。NEB已经成功克隆了180多种内切酶,其中大多是完全克隆,少数是部分克隆。目前,NEB可供应240多种内切酶,其中180多种可以重组酶形式提供,同时还有大量的应用途广泛的重组聚合酶和重组修饰酶。质量与客户服务NEB公司凭借着严格的质检程序、深入的基础科研以及不断开发的研发项目,承诺为全球科技人员提供高纯度的科研产品。直接与NEB总部联系或与NEB的国际网点联系,即可体验到NEB的个性化客户服务。公司内负责生产以及负责进行质量监控的科学家(产品负责人)也就是技术支持人员。他们为客户解答有关限制核酸内切酶、甲基化酶、以及其他DNA修饰酶、蛋白质修饰酶方面的问题。同时,公司的有机合成部门可提供linkers、引物、adaptors、探针以及寡核苷酸合成等方面的信息。研究人员也可为客户提供技术支持服务,这些研究人员在DNA测序、甲基化、克隆、过量表达、发酵、蛋白质纯化以及蛋白质分析方面都有很深造诣。基础科研NEB在分子生物学和寄生虫学方面的基础研究由公司内部的资深科学家负责。这些科学家在他们各自的领域都卓有成绩,经常在业内著名杂志上发表文献,指导博士后工作、为大学生提供暑期实习机会,而且经常受邀去当地学校进行演讲。NEB鼓励公司与外界合作,每周学术探讨会为其他科学家们提供了交流和展示的平台。环保政策NEB公司在资助科研项目时最基本原则之一就是:一切工作都应符合保护生态环境。公司产品生产、分析以及运输过程既要满足产品的稳定性,也要符合我们的环保政策。20年来,NEB公司最引人注目的一项环保政策是:回收运输泡沫盒,虽然这项工作首创于美国,但是,目前加拿大、德国以及英国的分公司也同样实施了。公司还在内部开展多方位的回收工作,经常用再生纸张印刷市场宣传资料。这些小细节不会影响产品质量,但是对环境的保护却是显而易见。公司职责除环保政策外,通过基金会,NEB还竭力为改善当地社区及全人类尽微薄之力。NEB基金会创立于1982年,属私人性质,其宗旨是支持发展中国家的环保、教育、健康及艺术事业。此外,NEB捐款委员会也经常向当地社区进行捐款活动。
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