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Magnesium chloride hexahydrate

氯化镁六水合物

Company: Sigma-Aldrich
Catalog#: M2670
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Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer
Author:
Date:
2020-05-05
[Abstract]  Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be compatible with advanced super-resolution techniques, mainly due to poor photophysical properties of their small-molecule fluorogens. Here, we describe a detailed protocol for fluorescence microscopy of mRNA in live bacteria using the recently reported fluorogenic silicon rhodamine binding aptamer (SiRA) featuring excellent photophysical properties. Notably, with SiRA, we demonstrated the first aptamer-based RNA visualization using super-resolution ... [摘要]  [摘要 ] 遗传编码的点亮适体是显示活细胞中RNA的有前途的工具,可帮助我们加深对RNA广泛而复杂的生命的理解。可见光波长区已经被开发,他们都没有然而,据报道,在兼容先进的超分辨率技术,主要是由于不良的光物理性质其小分子荧光团。在这里,我们描述了一个详细的协议对于荧光显微镜mRNA的使用最近报道的具有优异光物理性质的荧光罗丹明结合适体(SiRA )在活细菌中进行检测。值得注意的是,我们利用SiRA 展示了首个使用超分辨率(STED)显微镜进行的基于适体的RNA可视化。这种成像方法可能特别有价值用于可视化原核生物中的RNA,因为细菌的大小仅比光学分辨率大几倍 传统显微镜的分辨率。

[背景 ] 可视化的具体RNA分子通过荧光显微镜具有不可估量的价值在过去二十年中扩大我们的知识RNA功能内的细胞在时空精气神(特亚吉,2009年;夏等人,2017年),由于缺乏。固有的荧光RNA,用于活细胞成像的荧光RNA标记工具的开发以及它们对最新显微镜的适应性 –特别是对于超分辨率显微镜– 势在必行。超分辨率显微镜(SRM)对于原核系统中的RNA成像特别有吸引力,因为细菌很小(〜2.5MYU中号长,0.5-1〜MYU 中号宽)和分辨率的标准荧光显微镜被限制在200〜300〜牛米,由于衍射极限光(Reshes ...

A Highly Sensitive Anion Exchange Chromatography Method for Measuring cGAS Activity in vitro
Author:
Date:
2018-10-20
[Abstract]  Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor (PRR) that senses double stranded DNA (dsDNA) in the cytosol and this leads to the activation of stimulator of interferon genes (STING) via the secondary messenger 2’3’-cyclic GMP-AMP (2’3’-cGAMP). STING then recruits TANK binding kinase 1 (TBK-1) and this complex can phosphorylate and activate interferon regulatory factor 3 (IRF3) leading to the induction of type I interferons and other antiviral genes. The cGAS:DNA complex catalyzes the synthesis of 2’3’-cGAMP and the purpose of the protocol presented here is to measure the in vitro activity of purified cGAS in the presence of dsDNA. The protocol was developed to elucidate the relationship between dsDNA length and the level of cGAS activity. The method involves an in ... [摘要]  环状GMP-AMP合酶(cGAS)是一种模式识别受体(PRR),可以感知胞质溶胶中的双链DNA(dsDNA),并通过第二信使2'3'来激活干扰素基因刺激物(STING)。环GMP-AMP(2'3'-cGAMP)。然后STING募集TANK结合激酶1(TBK-1),该复合物可磷酸化并激活干扰素调节因子3(IRF3),导致I型干扰素和其他抗病毒基因的诱导。 cGAS:DNA复合物催化2'3'-cGAMP的合成,这里提出的方案的目的是测量在dsDNA存在下纯化的cGAS的体外>活性。开发该方案是为了阐明dsDNA长度与cGAS活性水平之间的关系。该方法涉及与低浓度cGAS和dsDNA的体外>反应,然后使用阴离子交换色谱法定量反应产物。当比较不同DNA片段激活cGAS的能力时,低浓度的cGAS和dsDNA以及该测定的高灵敏度是关键优势。

【背景】细胞胞质内存在双链DNA是DNA或逆转录病毒感染的潜在迹象。核苷酸转移酶cGAS作为感知细胞溶质dsDNA的模式识别受体起作用。 cGAS被dsDNA变构激活并催化ATP和GTP转化为环状二核苷酸2'3'-cGAMP(或简称cGAMP)(Ablasser et al。>,2013; Civril et al 。>,2013; Diner et al。>,2013; Gao et al。>,2013; ...

Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly
Author:
Date:
2018-08-05
[Abstract]  Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was ... [摘要]  染色体构象捕获测序(Hi-C)是一种全面询问细胞核中染色质三维定位的有效方法。 Hi-C的发展可以追溯到染色体构象捕获的分辨率和通量的连续增加(3C)(Dekker et al。,2002)。 3C的基本工作流程包括(i)通常用甲醛固定完整的染色质,(ii)用限制酶切割固定的染色质,(iii)在稀释条件下重新连接粘性末端,以促进交联片段之间的连接或随机片段之间的那些和(iv)量化基因组基因座对之间的连接事件的数量(de Wit和de Laat,2012)。在最初的3C方案中,通过半定量PCR扩增对应于少量基因组位点(“一对一”)的选定连接接头来测量连接频率(Dekker et al。,2002 )。然后,染色体构象捕获芯片(4C)和染色体构象捕获碳复制(5C)技术扩展3C以分别以“一对多”或“多对多”方式计算结扎事件。 Hi-C(Lieberman-Aiden et al。,2009)最终将3C与下一代测序相结合(Metzker,2010)。此处,在再连接之前,用生物素标记的核苷酸类似物填充粘性末端以在后续步骤中富集具有连接连接的片段。然后对Hi-C文库进行高通量测序,并将得到的读数映射到参考基因组,允许以“多对多”方式确定接触概率,其分辨率仅受限制性位点的分布限制和阅读深度。 Hi-C的首次应用是阐明人类基因组中的全球染色质折叠原理(Lieberman-Aiden et ...

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