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Company: Sigma-Aldrich
Catalog#: S0266
Other protocol()

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 ...

MNase Digestion for Nucleosome Mapping in Neurospora
[Abstract]  Digestion of chromatin by micrococcal nuclease MNase followed by high throughput sequencing allows us to determine the location and occupancy of nucleosomes on the genome. Here in this protocol we have described optimized conditions of MNase digestion of filamentous fungus Neurospora crassa chromatin without a requirement of a nuclear fractionation step. [摘要]  通过微球菌核酸酶MNase消化染色质,然后高通量测序允许我们确定核小体在基因组上的位置和占据。 在这个协议中,我们描述了MNase消化丝状真菌粗糙链孢霉染色体的优化条件,而不需要核分馏步骤。

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)。