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Acetic acid

醋酸

Company: Sigma-Aldrich
Catalog#: 695092
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Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs
Author:
Date:
2018-09-05
[Abstract]  The physical properties of viral-length polyuridine (PolyU) RNAs, which cannot base-pair and form secondary structures, are compared with those of normal-composition RNAs, composed of comparable numbers of each of A, U, G and C nucleobases. In this protocol, we describe how to synthesize fluorescent polyU RNAs using the enzyme polynucleotide phosphorylase (PNPase) from Uridine diphosphate (UDP) monomers and how to fractionate the polydisperse synthesis mixture using gel electrophoresis, and, after electroelution, how to quantify the amount of polyU recovered with UV-Vis spectrophotometry. Dynamic light scattering was used to determine the hydrodynamic radii of normal-composition RNAs as compared to polyU. It showed that long polyU RNAs behave like linear polymers for which the radii scale ... [摘要]  将不能碱基配对并形成二级结构的病毒长度聚尿苷(PolyU)RNA的物理性质与正常组成RNA的物理性质进行比较,正常组成RNA由相当数量的A,U,G和C核碱基组成。 在该协议中,我们描述了如何使用来自二磷酸尿苷(UDP)单体的酶多核苷酸磷酸化酶(PNPase)合成荧光polyU RNA以及如何使用凝胶电泳分离多分散合成混合物,并且在电洗脱后,如何量化 用UV-Vis分光光度法回收polyU。 与polyU相比,动态光散射用于确定正常组成RNA的流体动力学半径。 结果表明,长polyU RNA的行为类似于线性聚合物,其半径范围为链长为N 1/2 ,而正常组成RNA则作为紧凑的支链RNA,其半径范围为 如N 1/3 。

【背景】 PolyU作为物理对象: PolyU是一种由重复的尿苷残基组成的非生物RNA分子,因此,它不能与Watson-Crick碱基对缺乏RNA二级结构(Martin和Ames,1962; Richards et al。,1963)。 PolyU具有非常弱的碱基堆积能量,导致缺乏螺旋排序 - 除了低于4°C(Richards et al。,1963)。由于缺乏这种结构,polyU ...

Formaldehyde Fixation of Extracellular Matrix Protein Layers for Enhanced Primary Cell Growth
Author:
Date:
2017-07-05
[Abstract]  Coating tissue culture vessels with the components of the extracellular matrix such as fibronectin and collagens provides a more natural environment for primary cells in vitro and stimulates their proliferation. However, the effects of such protein layers are usually rather modest, which might be explained by the loss immobilized proteins due to their weak non-covalent association with the tissue culture plastic. Here we describe a simple protocol for a controlled fixation of fibronectin, vitronectin and collagen IV layers by formaldehyde, which substantially enhances the stimulation of primary cell proliferation by these extracellular proteins. [摘要]  用诸如纤连蛋白和胶原的细胞外基质的组分涂覆组织培养容器为体外原代细胞提供更自然的环境并刺激它们的增殖。 然而,这种蛋白质层的作用通常相当适中,这可能由于与组织培养塑料的非共价结合弱而被固定蛋白质损失所解释。 在这里我们描述一个简单的协议,通过甲醛控制纤维连接蛋白,玻连蛋白和胶原IV层的固定,这大大增强了这些细胞外蛋白对原代细胞增殖的刺激。
【背景】细胞外基质(ECM)如纤连蛋白,层粘连蛋白,玻连蛋白和胶原蛋白的组分通常用于包被组织培养容器,因为它们为体外原代细胞提供更自然的环境并刺激它们的增殖( Sawada等人,1987; ...

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; ...

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