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PDS-1000/He TM System

PDS-1000 / He系统

Company: Bio-Rad Laboratories
Catalog#: 1652257
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Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions
[Abstract]  Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss. [摘要]  最近为三角褐指藻(Phaeodactylum tricornutum)和海绵假丝酵母(Thalassiosira pseudonana)建立了硅藻基因组编辑。 目前的协议,虽然开发的 T。 pseudonana ,可以修改编辑任何硅藻基因组,因为我们利用灵活,模块化的金门克隆系统。 主要步骤包括如何设计构建使用金门克隆靶向两个网站,允许一个精确的删除被引入目标基因。 解释转化方案,以及使用带移位测定和/或限制性位点丢失进行筛选的方法。

【背景】CRISPR-Cas正在迅速成为分子研究的一个关键方法。基于在细菌和古细菌中发现的病毒防御机制,CRISPR-Cas诱导基因组中精确位置的双链断裂(DSBs)。它涉及使用与CRISPR ...

CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum
[Abstract]  The establishment of the CRISPR/Cas9 technology in diatoms (Hopes et al., 2016; Nymark et al., 2016) enables a simple, inexpensive and effective way of introducing targeted alterations in the genomic DNA of this highly important group of eukaryotic phytoplankton. Diatoms are of interest as model microorganisms in a variety of areas ranging from oceanography to materials science, in nano- and environmental biotechnology, and are presently being investigated as a source of renewable carbon-neutral fuel and chemicals. Here we present a detailed protocol of how to perform CRISPR/Cas9 gene editing of the marine diatom Phaeodactylum tricornutum, including: 1) insertion of guide RNA target site in the diatom optimized CRISPR/Cas9 vector (pKS diaCas9-sgRNA), 2) ... [摘要]  在硅藻(Hopes ,2016; Nymark等人,2016)中建立了CRISPR / Cas9技术,使得能够简单,廉价和有效地引入目标 这个非常重要的真核浮游植物群的基因组DNA的改变。 硅藻在纳米和环境生物技术领域从海洋学到材料科学,各种领域的示范性微生物都是有意义的,目前正在作为可再生碳中和燃料和化学品的来源进行调查。 在这里,我们提出了如何进行海洋硅藻三角褐指藻CRISPR / Cas9基因编辑的详细方案,包括:1)在硅藻优化的CRISPR / Cas9载体(pKS diaCas9)中插入引导RNA靶位点 -sgRNA),2)用于将pKS diaCas9-sgRNA质粒导入P的生物弹道转化。 三分支毛细胞和3)基于高分辨率熔融的PCR测定以筛选CRISPR / Cas9诱导的突变。
【背景】CRISPR / Cas9系统已被证明是许多真核生物中非常有效和成功的基因组编辑系统,现在也包括微藻(Hopes等人,2016; Nymark等人)。 ,2016; Shin 等人,2016)。 CRISPR / Cas9系统包括引导RNA(gRNA)和称为Cas9的核酸酶(Sander and Joung,2014)。 这两个分子形成复合物,其中gRNA将复合物引导至感兴趣的靶标。 ...

Biolistic Bombardment for Co-expression of Proteins Fused to YFP and mRFP in Leaf Epidermal Cells of Phaseolus vulgaris ‘Red Mexican’
[Abstract]  Biolistic bombardment is based on coating of tungsten or gold particles with DNA and delivery of these “biobullets” into living plant cells under high pressure (Sudowe and Reske-Kunz, 2013). This method enables transient expression of a DNA construct encoding fusion of the protein of interest to a fluorescence protein e.g. GFP for microscopic approaches. Usually it is performed for plants for which infiltration with Agrobacterium tumefaciens does not work efficiently e.g. model plant Arabidopsis thaliana (Ueki et al., 2009). Although transfection rate is relatively low, it is still sufficient to analyze subcellular localization of the protein of interest under a fluorescence microscope. Here we present the protocol that was optimized for Nicotiana ... [摘要]  生物轰击基于用DNA涂覆钨或金颗粒,并在高压下将这些"生物小球"递送到活的植物细胞中(Sudowe和Reske-Kunz,2013)。 该方法使得能够瞬时表达编码感兴趣的蛋白质与荧光蛋白例如GFP的融合物的DNA构建体用于显微方法。 通常,其对于用土壤杆菌根瘤土壤渗入不能有效工作的植物进行,例如模拟植物拟南芥(Ueki et al。 ,2009)。 虽然转染率相对较低,但仍然足以在荧光显微镜下分析目标蛋白的亚细胞定位。 在这里,我们提出针对 Nicotiana benthamiana 优化的方案,并成功地应用于 Phaseolus vulgaris (Giska ,2013)。