Name: fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity
Uploaded: 2012-04-13T12:00:00
Description: Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

我们承认，化学，地质和生物科学部，办公室基础能源科学，科学办公室，美国能源部（奖DE-FG02-91ER20021）和国家科学基金会（MCB 0948584）（FB）的支持。我们感谢编辑手稿女士卡伦鸟的。

1。 EMS处理 拟南芥种子的诱变诱变剂乙基甲烷磺酸盐（EMS），3,4，这将导致基因组的C-T的变化的C / G / A突变5-7使用。 <ol><li>权衡0.8Ğ拟南芥种子携带的细胞器荧光标记（特别是，在这项研究中，ssGFPHDEL（信号序列的GFP-HDEL四肽）已作为ER标记）（〜40,000种子）。 </li><li>在50毫升的猎鹰管转移的种子，然后加入25毫升蒸馏水。 </li><li>添加0.2％（V / V）乙基...

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在这里，我们描述了共聚焦显微镜为基础的筛选为内膜突变体鉴定。这种方法可以很容易地扩展到其他细胞的细胞器，为特定的荧光蛋白标记。基于屏幕的鉴定表明，无论是在目标器官或细胞器，不应该包含标记荧光标记的异常分布的突变。 ，这些突变体分别代表人群中受到损害的器官的能力subcompartimentalize标记或突变体不能正常转运细胞器之间的标记。在筛选中，我们注意到，一些突变表明，?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> ethylmethane磺酸盐</td><td>西格玛</td><td> M0880 </td></tr><tr><td>氢氧化钠</td><td>托德·贝克</td><td> 3722-05 </td></tr><tr><td> murashige斯库格培养基的瓦特gamborg维生素</td><td>植物技术的laboratorie </td><td> M404 </td></tr><tr><td> phytagel </td><td>西格玛</td><td> P8169-1千克</td></tr><tr><td>的RNeasy迷你套件</td><td> Qiagen公司</td><td> 74104 </td></tr><tr><td>大师纯植物叶片DNA纯化试剂盒</td><td>震中</td><td> MPP92100 </td></tr><tr><td> bioprime的DNA标签制度</td><td> Invitrogen公司</td><td> 18094-011 </td></tr><tr><td>酒精200证明</td><td> decan实验室公司。 </td><td> 2716 </td></tr><tr><td>醋酸钠</td><td>托德·贝克</td><td></td></tr><tr><td>基因芯片拟南芥ATH1基因组阵列</td><td> Affymetrix公司</td><td> 900385 </td></tr><tr><td>猎鹰管50毫升</td><td>康宁</td><td> 430290 </td></tr><tr><td> Eppendorf管1.5毫升</td><td></td><td></td></tr><tr><td>滤波纸90毫米</td><td>瓦特曼</td><td> 1001090 </td></tr><tr><td>分析天平</td><td>梅特勒 - 托利多AB54-S的</td><td> NA </td></tr><tr><td> nutating（波）振动筛</td><td> heidolph普力马1040 </td><td> NA </td></tr><tr><td>离心分离</td><td> Eppendorf公司5417 - R </td><td> NA </td></tr></tbody></table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

本协议描述了拟南芥幼苗的荧光显微镜为基础的筛选，并介绍如何将隐性突变，改变了一个特定的标记分泌途径中的荧光标记的亚细胞分布。 拟南芥是一种强大的生物遗传研究模型，因为它的基因组大小，一代的时间，并节约王国之间的分子机制。作为一种方法来映射替代传统的方法，基于分子标记的突变基因分型阵列是有利的，因为它是相对较快，可能允许在一个非常短的时间框架的几个突变的映射。这种方法允许蛋白质的鉴定，可以影响任何在植物细胞器的完整性。在这里，作为一个例子，我们提出了一个重要的内质网（ER）的完整性的地图基因的屏幕。然而，我们的做法，可以很容易地扩展到其他植物细胞器（例如，见1,2），因此代表了对理解其他亚细胞结构的分子基础的重要一步。

Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

在细胞生物学的一个基本任务是定义机制的基础，使真核细胞的细胞器的身份。在这里，我们提出了一个方法，以确定该基因的植物细胞器，利用荧光显微镜和下一代测序工具的形态和功能的完整性负责。

荧光显微镜筛选和新一代测序：参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Research

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Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

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One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

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The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

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Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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Interactome: Domainome 图书馆建设、噬菌体展示和下一代测序的验证与选择协议

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