The phi29-XT RCA Kit includes everything needed for sensitive and robust rolling circle amplification (RCA), an isothermal amplification method to continuously amplify circular DNA by generating long, repetitive copies of the circular sequence. The kit features phi29-XT DNA Polymerase, an engineered polymerase with improved thermostability and sensitivity, and generates higher yieldin a shorter reaction time than wild-type phi29 DNA Polymerase. Also included are dNTPs and exonuclease-resistant random primers (containing phosphorothioate bonds) to universally amplify circular DNA sequences. Input material can be purified circular DNA (single or double-stranded) or direct from liquid media culture, agar plate colonies, and glycerol stocks without the need for DNA extraction. RCA products can be used directly in downstream applications such as DNA sequencing, cell-free DNA enrichment, cell-free protein expression, and DNA biosensors.Figure 1: Overview of the phi29-XT RCA KitThe phi29-XT RCA Kit (NEB #E1603) is a fast, simple to use, and highly versatile kit containing all the required components for rolling circle amplification (RCA) using a random primer mix. The kit delivers high yields of DNA products from a variety of starting materials including purified circular DNA or bacterial cells. This kit is ideal for various DNA applications such as DNA sequencing, cell-free DNA enrichment, cell-free protein expression and DNA biosensors.Figure 2: phi29-XT DNA Polymerase generates more product in less time than wild-type phi29 DNA Polymerase, at elevated temperaturesTriplicate RCA reactions were conducted with 0.1 pg pUC19 plasmid input (NEB #N3041) at the indicated temperatures for 2 hours, followed by heat inactivation at 65°C for 10 minutes. All reactions contained 1 mM dNTPs and 50 µM Exonuclease-Resistant Random Primers. Wild-type phi29 reactions were carried out with 10 units of phi29 DNA Polymerase (NEB #M0269) in 1X phi29 DNA Polymerase Reaction Buffer and 0.1 mg/mL Recombinant Albumin, and phi29-XT reactions were carried out with 1X phi29-XT DNA Polymerase and 1X phi29-XT Reaction Buffer. Reaction yields (dots) were quantified using Quant-iT® PicoGreen® dsDNA Reagent and were averaged (line) to determine the yield at each reaction temperature. While the working reaction temperature of wild-type phi29 DNA Polymerase is between 30°C and 37°C, phi29-XT DNA Polymerase generates robust product yields around 42°C.Figure 3: The phi29-XT RCA Kit efficiently amplifies as little as 1 fg pUC19 DNAQuadruplicate RCA reactions were carried out with the phi29-XT RCA Kit (NEB #E1603) at 42°C for 2 hours in the absence of template (NTC) or with 1 fg pUC19 plasmid. Reaction products were digested with AclI (NEB #R0598), which site-specifically cuts the plasmid sequence to generate two fragments (373 bp and 2,313 bp), and run on an agarose gel to verify successful amplification. phi29-XT DNA Polymerase supports robust amplification of 1 fg of 2.7 kb circular plasmid DNA in just 2 hours.Figure 4: The phi29-XT RCA Kit offers exceptional sensitivity and product yieldTriplicate RCA reactions were carried out using commercially available phi29 DNA polymerases, according to manufacturers’ protocols, for 2 hours with 1 pg or 1 ng pUC19 plasmid as the starting material. Reaction yields (dots) were quantified using Quant-iT®PicoGreen® dsDNA Reagent and averaged (bar). The phi29-XT RCA Kit (NEB #E1603) generates more product in less time than other commercially available products.Figure 5: Direct colony RCA with the phi29-XT RCA Kit enables Sanger sequencing andcell-free protein expression in less time than a traditional plasmid preparation approachA. A single bacterial colony, harboring a modified pET28a vector with eGFP under a T7 promoter, was picked and diluted in water. Additional plasmid was then produced in vitro using the phi29-XT RCA Kit (NEB #E1603), or by traditional liquid culture using bacterial cells.B. Sanger sequencing was performed on the RCA product (1 µl of 20-fold diluted RCA reaction) or plasmid (900 ng) extracted from the liquid culture, as depicted.C. Following sequence verification, GFP protein was expressed with the NEBExpress® Cell-free E. coli Protein Synthesis System (NEB #E5360) using either 2.4 µl of 20-fold diluted RCA reaction or 100 ng of purified plasmid in 10 µl reaction volume. Protein synthesis was monitored by measuring eGFP fluorescence, highlighting that RCA products enable robust cell-free protein expression of GFP in a shorter amount of time than plasmid prep.
This product is related to the following categories:
This product can be used in the following applications:
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。
