{{'Search' | translate}}
 

QIAquick PCR Purification Kit

QIAquick PCR纯化试剂盒

Company: QIAGEN
Catalog#: 28104
Bio-protocol()
Company-protocol()
Other protocol()

Design and Direct Assembly of Synthesized Uracil-containing Non-clonal DNA Fragments into Vectors by USERTM Cloning
Author:
Date:
2017-11-20
[Abstract]  This protocol describes how to order and directly assemble uracil-containing non-clonal DNA fragments by uracil excision based cloning (USER cloning). The protocol was generated with the goal of making synthesized non-clonal DNA fragments directly compatible with USERTM cloning. The protocol is highly efficient and would be compatible with uracil-containing non-clonal DNA fragments obtained from any synthesizing company. The protocol drastically reduces time and handling between receiving the synthesized DNA fragments and transforming with vector and DNA fragment(s). [摘要]  该协议描述如何通过基于尿嘧啶切除的克隆(USER克隆)命令并直接组装尿嘧啶非克隆DNA片段。 该方案的目的是制备与USER TM克隆直接兼容的合成非克隆DNA片段。 该协议是高效率,并将与从任何合成公司获得尿嘧啶非克隆DNA片段相容。 该方案大大减少了接收合成的DNA片段和用载体和DNA片段转化之间的时间和处理。

【背景】对于合成的DNA,非克隆线性DNA片段(NCDF)已经成为更廉价和更快速的替代克隆片段的方法,并且在循环载体中被测序。 NCDF可以被认为是IKEA DNA合成的解决方案,在这个解决方案中,客户将他们的DNA片段组装成一个选择的载体,随后必须验证最终构建体的序列。在这个协议中,我们从Thermo Fisher Scientific ...

Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli
Author:
Date:
2017-10-05
[Abstract]  We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by ... [摘要]  我们提出了构建大肠杆菌可调CRISPR干扰(tCRISPRi)菌株的方案。 tCRISPRi系统缓解了基于质粒的表达方法的大多数已知问题,并且可以立即用于构建可通过靶向必需基因和非必需基因来补充Keio收集物的sgRNA的文库。 最重要的是从实践的角度来看,建立tCRISPRi来靶向一个新的基因只需要一步寡核苷酸重组。 tCRISPRi与其他现有CRISPRI方法的其他优点包括:(1)tCRISPRi显示低于10%的泄漏抑制; (2)tCRISPRi使用可调阿拉伯糖操纵子启动子和转运蛋白基因的修饰,以允许通过阿拉伯糖诱导剂分级控制的宽动态范围; (3)tCRISPRi是无质粒的,整个系统整合到染色体中; (4)tCRISPRi菌株显示出理想的生理特性。
【背景】已经开发了各种CRISPR干扰系统,用于从细菌到真核生物的生物体。对于正在考虑使用CRISPRi细菌的人员,我们提供了关于我们的tCRISPRi系统的以下背景资料(Li等等,2016)及其与其他CRISPRi系统的比较。
Morgan-Kiss 等人。 (2002)开发了基于质粒的剂量诱导型启动子pBAD。它们的系统允许来自pBAD启动子的蛋白质的可调节表达,取决于阿拉伯糖水平。阿拉伯糖转运蛋白基因和araFGH在菌株中是无活性的。他们的菌株也有两个拷贝的lacY ...

Dense sgRNA Library Construction Using a Molecular Chipper Approach
Author:
Date:
2017-06-20
[Abstract]  Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. We present a Molecular Chipper protocol for generating dense sgRNA libraries from genomic regions of interest. This approach utilizes a combination of random fragmentation and a Type III restriction enzyme to derive a dense coverage of sgRNA library from input DNA. [摘要]  使用单导向RNA(sgRNA)文库和CRISPR技术的遗传筛选功能强大可以识别基因组编码区和非编码区的遗传调控因子。 在非编码区域中询问功能元件需要密集覆盖的sgRNA文库,理想的便宜,易于实现和灵活定制。 我们提出了一个分子切片方案从感兴趣的基因组区域产生密集的sgRNA文库。 该方法利用随机断裂和III型限制酶的组合从输入DNA导出sgRNA文库的致密覆盖。
【背景】使用化脓性链球菌(sp)的基因组编辑Cas9和sgRNA文库是通过产生双重缺失功能序列改变来筛选哺乳动物细胞功能性遗传调节因子的有力工具(Wiedenheft et al。,2012; Mali et al。,2013; Koike-Yusa等,2014; Shalem等,2014; Wang等,2014; Zhou等,2014)。 Cas9结合sgRNA,其可被设计为将Cas9靶向基因组中定义的基因座。 Cas9的核酸酶活性切割靶DNA位点,导致双链DNA断裂,在通过非同源末端连接途径进行DNA修复时,经常导致感兴趣的基因座短缺失。
CRISPR-Cas9系统强大的基因组编辑能力导致使用sgRNA文库来询问蛋白质编码基因以及非编码区域。通过sgRNA富集功能筛选,报告了几种用于蛋白质编码基因和/或有限数量的非编码基因的sgRNA文库,以鉴定调控特定细胞功能的基因和网络(Koike-Yusa等,2014; ...

Comments