The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is designed to provide a simple, one-step solution for LAMP/RT-LAMP of DNA or RNA targets. LAMP and RT-LAMP are commonly used isothermal amplification techniques that provide rapid detection of a target nucleic acid using LAMP-specific primers (supplied by the user) and a strand-displacing DNA polymerase. This master mix features a blend of Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx Reverse Transcriptase in an optimized LAMP buffer solution. Both Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx Reverse Transcriptase have been engineered for improved performance in LAMP and RT-LAMP reactions. The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is fully buffered and compatible with different sample types, enabling multiple detection methods including turbidity detection, real-time fluorescence detection (when used with LAMP fluorescent dye) and end-point visualization such as colorimetric detection via a metal indicator (e.g., hydroxynaphthol blue.)The inclusion of dUTP and thermolabile UDG in the mix reduces the possibility of carryover contamination, where unintended product of a previous amplification can serve as the substrate of a subsequent reaction. Thermolabile UDG is completely inactivated at temperatures above 50°C, thereby having no effect on the reaction.Figure 1: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is compatible with multiple detection methodsAmplification of target RNA or DNA by the WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) (NEB #M1708) can be confirmed using a variety of real-time (A) and endpoint (B) detection methods. Some of these detection modalities include fluorescence, non-pH-based colorimetric (e.g., hydroxynaphthol blue), turbidity or agarose gel electrophoresis. For real-time fluorescence detection, the master mix is available as a kit (NEB #E1708) that includes 50X LAMP Fluorescent Dye, which can be monitored in the SYBR®/FAM channel of most real-time PCR instruments.Figure 2:LAMP/RT-LAMP master mixes and sample type considerationsNEB’s pH-based colorimetric LAMP master mixes with UDG (NEB #M1804) or without UDG (NEB# M1800) are weakly buffered to allow for visual detection of amplification using a pH-sensitive dye. However, the low buffering capacity required to generate the pink to yellow color change limits sample compatibility with the pH-based colorimetric mixes, as highly buffered sample inputs or acidic samples may impact the color change. The multi-purpose LAMP/RT-LAMP 2X Master Mix with UDG (NEB #M1708, NEB #E1708) or without UDG (NEB #E1700) is fully buffered and can more readily tolerate these types of sample inputs, making it compatible with various detection modes including fluorescence or other colorimetric dyes (e.g., hydroxynaphthol blue).Figure 3: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) provides robust detection of human DNA and RNA targetsRT-LAMP (RNA targets) or LAMP (DNA targets) experiments were performed with NEB #M1700: WarmStart LAMP 2X Master Mix, which is the master mix in the NEB #E1700kit that does not contain dUTP/UDG, and NEB #M1708: WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG). Reactions containing 1X LAMP primers and 1X LAMP Fluorescent dye were set up in quadruplicate over three logs of total Jurkat RNA or Jurkat DNA (10 ng to 0.1 ng) in 96-well, 25 µl reactions. Control reactions without template (NTC) were also evaluated. Reactions were incubated at 65°C for 40 minutes and fluorescence was monitored every 15 seconds in the SYBR/FAM channel of a real-time thermocycler (Bio-Rad® CFX96). Each dot represents the time at which the fluorescence signal for a single reaction crosses the instrument-defined threshold. All four replicates were detected at each template input unless otherwise indicated (note that dots frequently overlap given similar detection time for the replicates). Overall, similar performance was observed for both NEB #M1700and NEB #M1708 at each template input. No amplification was observed in any of the no template control reactions.Figure 4: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is compatible with automated reaction assemblyLAMP assays targeting synthetic SARS-CoV-2 RNA were assembled either manually or with the TEMPEST® liquid handling platform (96-well, 25 µl reactions). The assay was carried out using either positive samples (human total RNA plus synthetic SARS-CoV-2 RNA at 5,000, 500 or 50 copies per reaction) or no template (NTCs) as indicated. Reactions were incubated at 65°C for 40 minutes and monitored with 1X LAMP Fluorescent dye in the SYBR/FAM channel of a real-time instrument (Bio-Rad CFX96). Similar time to detection and LOD results were observed for both reaction assembly methods.Figure 5: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is compatible with non-pH-based colorimetric detection (e.g., hydroxynaphthol blue)The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) was used to amplify synthetic SARS-CoV-2 RNA using a dual primer mix that targets the N and E genes in the presence of 0.12 mM of hydroxynaphthol blue, a colorimetric metal indicator. The assay was carried out using either positive samples (human total RNA plus synthetic SARS-CoV-2 RNA at 5,000, 500 or 50 copies per reaction), negative samples (human total RNA alone) or no template (NTCs), as indicated. The 25 µl reactions were incubated at 65°C for 45 minutes and then visually inspected.A. All positive samples gave a positive result with robust color change from purple to blue, including detection in all samples at a low input of 50 copies per reaction (n = 20).B. No color change was observed for numerous replicates of negative samples (n = 32).
This product is related to the following categories:
This product can be used in the following applications:
Loop-Mediated Isothermal Amplification
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。
