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One ShotTM TOP10 Chemically Competent E. coli

Company: Thermo Fisher Scientific
Catalog#: C404010
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Dual Fluorescence Reporter Based Analytical Flow Cytometry for miRNA Induced Regulation in Mammalian Cells
Author:
Date:
2018-09-05
[Abstract]  MicroRNA-induced gene regulation is a growing field in basic and translational research. Examining this regulation directly in cells is necessary to validate high-throughput data originated from RNA sequencing technologies. For this several studies employ luciferase-based reporters that usually measure the whole cell population, which comes with low resolution for the complexity of the miRNA-induced regulation. Here, we provide a protocol using a dual-fluorescence reporter and flow cytometry reaching single cell resolution; the protocol contains a simplified workflow that includes: vector generation, data acquisition, processing, and analysis using the R environment. Our protocol enables high-resolution measurements of miRNA induced post-transcriptional gene regulation and combined with ... [摘要]  MicroRNA诱导的基因调控是基础和转化研究中不断增长的领域。 直接在细胞中检查该调节对于验证源自RNA测序技术的高通量数据是必要的。 对于这一研究,一些研究采用基于荧光素酶的报告基因,通常测量全细胞群,其具有低分辨率的miRNA诱导调节的复杂性。 在这里,我们提供使用双荧光报告基因和流式细胞仪达到单细胞分辨率的方案; 该协议包含一个简化的工作流程,包括:使用R环境进行矢量生成,数据采集,处理和分析。 我们的协议可实现miRNA诱导的转录后基因调控的高分辨率测量,并结合系统生物学,可用于估计miRNA的熟练程度。

【背景】MicroRNAs(miRNA)是高度保守的小型非蛋白质编码RNA(21-22nt),可调节转录后基因表达并调节基因生物学过程,如发育和细胞稳态(Lagos-Quintana et al。,2001; Fabian et al。,2010; Bartel,2018),包括miRNA表达与肿瘤进展和侵袭性相关的几种病理(Lu et al。, 2005; Di Leva和Croce,2013; Krishnan et al。,2015; Bertoli et ...

Single and Multiplexed Gene Editing in Ustilago maydis Using CRISPR-Cas9
Author:
Date:
2018-07-20
[Abstract]  The smut fungus Ustilago maydis is an established model organism for elucidating how biotrophic pathogens colonize plants and how gene families contribute to virulence. Here we describe a step by step protocol for the generation of CRISPR plasmids for single and multiplexed gene editing in U. maydis. Furthermore, we describe the necessary steps required for generating edited clonal populations, losing the Cas9 containing plasmid, and for selecting the desired clones. [摘要]  黑穗病真菌 Ustilago maydis 是一种既定的模式生物,用于阐明生物营养病原体如何在植物中定殖以及基因家族如何对毒力作出贡献。 在这里,我们描述了用于生成用于 U中单个和多重基因编辑的CRISPR质粒的逐步方案。玉米小斑病。 此外,我们描述了产生编辑的克隆群体,丢失含有Cas9的质粒以及选择所需克隆所需的必要步骤。

【背景】担子菌真菌 U. maydis 是一种模式生物,能够在DNA修复和同源重组,交配,性发育,次级代谢,丝状生长,RNA转运和毒力等主题中获得重要发现(Holliday,2004; Steinberg,2007; Bakkeren et al。,2008; Holloman et al。,2008; Lanver et al。,2017; Niessing et al。 ,2018年)。 Ú。 maydis 是一种生物营养的植物病原体,可引起玉米的黑穗病。作为大群黑穗病真菌的典型成员,其性发育与其定殖植物的能力密切相关。 U的受欢迎程度。 maydis 作为一种模式生物存在于其短暂的致病生命周期中,该生命周期在不到两周内完成,其可用于正向和反向遗传,包括建立自我复制的质粒(Tsukuda 等。,1988),能够在没有交配的情况下引起疾病的致病性病毒株的开发和可用的注释良好的基因组(Kämper et al。,2006)。在 ...

Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis
Author:
Date:
2018-02-05
[Abstract]  Chlamydomonas reinhardtii is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann et al., 1999; Schroda et al., 2000; Schroda, 2006). Among them, forward genetic approach has been intensively used, mostly because of the high efficiency in the generation of hundreds of thousands of mutants by random insertional mutagenesis and the haploid nature therefore phenotypic analysis can be done in the first generation (Cagnon et al., 2013; Tunçay et al., 2013). A major bottleneck in the application of high throughput methods in a forward genetic approach is the identification of the genetic lesion(s) responsible for the ... [摘要]  莱茵衣藻(Chlamydomonas reinhardtii)是光合作用,代谢和鞭毛生物学的基础研究者。已经为这种藻类开发了多功能的工具箱(Fuhrmann等人,1999; Schroda等人,2000; Schroda,2006)。其中,正向遗传方法已经被广泛使用,主要是因为通过随机插入诱变产生了数十万个突变体的高效率,并且可以在第一代进行单倍体性质表型分析(Cagnon等人 2013,Tunçay et。 2013)。在正向遗传方法中应用高通量方法的主要瓶颈是鉴定与观察到的表型相关的遗传损伤。在该协议中,我们详细描述了最初在(González-Ballester等人,2005)中报道的限制性定点扩增PCR(RESDA-PCR)的改进版本。优化包括引物组合的优化,DNA聚合酶的选择,PCR循环参数的优化以及PCR产物直接测序的应用。这些修改使得获得特定的PCR产物变得更加容易,并且加速了亚克隆步骤以更快地获得测序数据。


【背景】除了限制酶位点定向扩增PCR(RESDA-PCR)(González-Ballester等人,2005)之外,还发现了其它几种分子技术。 包括Genome ...

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