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Surgical tape, MicroporeTM 1530


Company: VWR
Catalog#: 115-8172
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Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9
[Abstract]  Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional regulation, has been published. So far, the generation of multiple mutants in plants involved tedious crossing or mutagenesis followed by time-consuming screening of huge populations and the use of the Cas9-system appeared a promising method to overcome these issues. We designed a binary vector that combines both the coding sequence of the codon optimized Streptococcus pyogenes Cas9 nuclease under the control of the Arabidopsis thaliana ... [摘要]  自从发现CRISPR(聚集的定期交织的短回文重复) - 相关蛋白(Cas)作为植物基因组编辑的有效工具(Li等人,2013; Shan等人已经出版了诸如基因敲除,敲入或转录调控等各种各样的应用,例如,2013; Nekrasov等人,2013)。到目前为止,植物中多种突变体的产生涉及繁琐的杂交或诱变,随后大量人群的耗时筛选,Cas9系统的使用似乎是有希望的方法来克服这些问题。我们设计了一种二元载体,其结合了在拟南芥UBIQUITIN10(UBQ10)启动子和引导RNA(gRNA)控制下的优化的化脓性链球菌(Caspase)密码子的编码序列)由 A驱动的表现盒。拟南芥U6 - 启动子,用于在拟南芥中进行有效的多重编辑(阎等人,2016年)。在这里,我们描述了一个逐步的方案,以经济有效的方式生成含有多个gRNA的二元载体和基于经典克隆方法的Cas9核酸酶。背景 RNA引导的Cas9系统源于针对外源DNA的细菌防御系统(Sorek等人,2013)。由于其高效率,易于处理和多重编辑的可能性,已经被认为是基因组编辑的选择方法。通常,Cas9基因编辑系统涉及单个合成RNA分子,其指导Cas9蛋白质靶向所需DNA位点以进行基因组修饰或转录控制的gRNA。 gRNA-Cas9复合物通过gRNA-DNA配对识别靶向的DNA,并需要存在原始相邻基序(PAM)。 ...

Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds
[Abstract]  The Arabidopsis thaliana seed coat epidermis produces copious amounts of mucilage polysaccharides (Haughn and Western, 2012). Characterization of mucilage mutants has identified novel genes required for cell wall biosynthesis and modification (North et al., 2014). The biochemical analysis of seed mucilage is essential to evaluate how different mutations affect cell wall structure (Voiniciuc et al., 2015c). Here we describe a robust method to screen the monosaccharide composition of Arabidopsis seed mucilage using ion chromatography (IC). Mucilage from up to 48 samples can be extracted and prepared for IC analysis within 24 h (only 4 h hands-on). Furthermore, this protocol enables fast separation (31 min per sample), automatic detection and ... [摘要]  拟南芥种皮表皮产生大量的粘液多糖(Haughn和Western,2012)。 粘液突变体的表征已经鉴定了细胞壁生物合成和修饰所需的新基因(North等人,2014)。 种子粘液的生物化学分析对于评估不同突变如何影响细胞壁结构是必要的(Voiniciuc等人,2015c)。 在这里我们描述了使用离子色谱(IC)筛选拟南芥种子粘液的单糖组成的有力方法。 在24小时内(仅4小时手动)可以提取并准备来自多达48个样品的粘蛋白并进行IC分析。 此外,该协议使快速分离(每个样品31分钟),自动检测和定量的中性和酸性糖。

Localisation and Quantification of Reactive Oxygen Species and Nitric Oxide in Arabidopsis Roots in Response to Fungal Infection
[Abstract]  Nitric oxide and reactive oxygen species have emerged as important signalling molecules in plants. The half-lives of NO and ROS are very short therefore rapid and precise measurements are required for the understanding biological roles of these redox active species. Various organelles and compartments generate NO and ROS thus it is important to determine precise location of these free radicals in order to understand their signalling roles. Diaminofluorescen (DAF) and fluorescent 2', 7'-dichlorofluorescein (DCF) dyes are employed to determine NO and ROS localisation. The advantage of this approach is that the dyes diffuse precisely to NO and ROS producing sites and generate fluorescence which can be detected by fluorescence- or confocal laser scanning microscopes. However, this technique ... [摘要]  一氧化氮和活性氧在植物中作为重要的信号分子出现。 NO和ROS的半衰期非常短,因此需要快速和精确的测量来理解这些氧化还原活性物质的生物学作用。各种细胞器和隔室产生NO和ROS,因此重要的是确定这些自由基的精确位置,以了解他们的信号传导作用。使用二氨基荧光素(DAF)和荧光2',7'-二氯荧光素(DCF)染料来确定NO和ROS定位。这种方法的优点是染料精确扩散到NO和ROS产生位点,并产生可以通过荧光或共聚焦激光扫描显微镜检测的荧光。然而,这种技术有其缺点;特别是需要建立荧光信号的特异性。因此,需要诸如cPTIO和抗坏血酸的ROS的使用清除剂来确认荧光信号的特异性,并且理想地,由于与每种方法相关的优点和缺点,理想地确认使用其它方法获得的数据(Gupta和Igamberdiev,2013 )。在这里我们描述了一种方法来检测响应于木霉属,镰刀菌属使用DAF,气相Griess试剂测定和DCF荧光方法的感染的拟南芥根的NO和ROS产生。