Intended for RT-PCR, the LunaScript Multiplex One-Step RT-PCR Kit offers a streamlined protocol for cDNA synthesis and PCR amplification in a single reaction. The 5X reaction mix contains dNTPs and is optimized for multiple target detection in a simple workflow. The 25X enzyme mix features Luna WarmStart Reverse Transcriptase and Q5 Hot Start High-Fidelity DNA Polymerase. The dual-temperature control of enzyme activities by aptamer-based inhibition enables room temperature reaction setup, with preassembled reactions stable at room temperature for up to 24 hours. The kit has robust multiplex target amplification capacity and enables various applications such as diagnostics, pathogen detection, and viral genome sequencing (including the ~50 amplicons per reaction used in ARTIC SARS-CoV-2 sequencing protocols).Multiplex RNA target detection and identification from a single RT-PCR reactionThe LunaScript Multiplex One-Step RT-PCR Kit (NEB #E1555) requires only RNA template and gene-specific primers to enable multiplex cDNA target synthesis and amplification in a single reaction. Amplified cDNA products can be detected or identified by downstream applications including next-generation sequencing, DNA arrays, fragment analysis, electrophoresis, and traditional cloning/sequencing workflows.Superior multiplexing and sensitivity with the LunaScript Multiplex One-Step RT-PCR KitHuman spleen total RNA was used to amplify 7 targets ranging from 185–776 bp using the LunaScript Multiplex One-Step RT-PCR Kit (NEB #E1555) or the SuperScript IV One-Step RT-PCR System (Thermo Fisher Scientific). 40% of each RT PCR experiment was loaded onto a 2% agarose gel. For LunaScript reactions, the standard protocol of 1 μM final primer concentration and a 1 minute annealing step was used. For the SSIV reactions, the recommended conditions failed to amplify all bands. Additional conditions were tested, varying key parameters of primer concentration and annealing time. The LunaScript Multiplex One-Step RT-PCR Kit successfully amplified all 7 targets from a variety of RNA inputs, and no conditions were identified that enabled the SuperScript IV One-Step RT-PCR System to amplify the full spectrum of targets, even at high input.The LunaScript Multiplex One-Step RT-PCR Kit enables simple ARTIC SARS-CoV-2 sequencing workflowsAmplicons were generated from 1000 copies of SARS-CoV-2 viral gRNA (ATCC VR-1986) in 100 ng of Universal Human Reference RNA (Thermo Fisher Scientific®, QS0639) using LunaScript Multiplex One-Step RT-PCR Kit with ARTIC V3 primers from either NEB (A) or IDT (B). cDNA synthesis was conducted in duplicate 12.5 μl reactions for each primer pool (1 and 2) using the following cycling conditions: 20 min at 55°C (reverse transcription), 1 min at 98°C (RT inactivation/initial denaturation), followed by 35 cycles of 15 sec at 95°C and 5 min at 63°C (annealing and extension).Libraries were constructed from 1.3 μl of each sample of pooled amplicons using the NEBNext® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®) and sequenced on a MiSeq® instrument (2X75 bp). Sequencing reads per amplicon were plotted for each library. Reads were downsampled with seqtk (250,000 read pairs) and aligned to the SARS-CoV-2 reference genome (NCBI, NC_045512) with Bowtie 2. Reference line indicates typical quality threshold of 100 reads.Sensitive and robust detection of RNA with up to 24 hours of room temperature stabilityA 570 bp GAPDH target was amplified from 10 ng down to 0.01 pg of Jurkat total RNA using the LunaScript Multiplex One-Step RT-PCR Kit (NEB #E1555). Reactions were set up on ice and run immediately (Control) or were set up and left at room temperature for 1, 4 or 24 hours before thermocycling. No template control (NTC) reactions were set up for each incubation condition. Highly sensitive and specific target amplification was observed at all input amounts, even after the preassembled reactions were left at room temperature for up to 24 hours.Comparing the LunaScript and OneTaq one-step RT-PCR products
This product is related to the following categories:
RT-PCR Products,
Reverse Transcriptases & RT-PCR Products,
cDNA Synthesis & Reverse Transcriptases Products
This product can be used in the following applications:
RT-PCR,
cDNA Synthesis,
RT-PCR & cDNA Synthesis
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。
