Name: high-throughput yeast plasmid overexpression screen
Uploaded: 2011-07-27T13:00:00
Description: Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

这项工作是由惠普ALS研究中心的约翰霍普金斯（ADG），美国国立卫生研究院主任的新的创新奖1DP2OD004417 - 01（ADG），美国国立卫生研究院R01 NS065317（ADG），丽塔艾伦基金会学者奖授予的支持。助理总干事，是由皮尤慈善信托基金支持的“皮尤研究中心的学者，在生物医学科学”。

1。酵母转化的筹备工作此协议是专为10个96孔板，但可以向上或向下扩展。我们发现，这个协议不超过二96孔盘每一轮的转型工作。整个改造过程（步骤I.3），将需要约8小时。 <ol><li>分装到每一个圆底96孔板Biorobot RapidPlate液体处理程序以及FLEXGene从酵母的ORF库的质粒DNA（为100 ng /μL）5 -10μL。放置在洁净工作台，通风柜一夜之间干破获96孔板。我们已经发现类似与CEN和2μ酵母?...

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在这里，我们提出了一个协议来执行一种高通量质粒在酵母中的过度表达屏幕。这种方法可以让许多不同的细胞表型的遗传修饰词的迅速和公正的筛选。使用这种方法，研究人员可以在几个星期内屏幕的酵母基因组中的一个重要部分。这种不偏不倚的方式也可以修饰词的识别，预测基于以前的研究结果可能没有被。我们已经用这种方法来确定与人类神经退行性疾病的蛋白质的聚集 19,21-23?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen公司</td><td> 9000490 </td></tr><tr><td> 96螺栓复制（青蛙过河） </td><td> V＆P科学</td><td> VP404 </td></tr><tr><td> FLEXGene ORF的图书馆</td><td>蛋白质组学研究所，哈佛医学院</td><td></td></tr><tr><td>台式离心机</td><td> Eppendorf公司</td><td> 5810R </td></tr><tr><td> 500毫升莫名其妙烧瓶</td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L三重莫名其妙费恩巴赫烧瓶</td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100μLRapidplate枪头</td><td>爱思进</td><td> ZT - 100 - RS </td></tr><tr><td> 200μLRapidplate枪头</td><td>爱思进</td><td> ZT - 200 - RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

芽殖酵母， 酿酒酵母 ，是一个强大的模型系统，用于定义许多重要的细胞过程，包括那些与人类疾病直接相关的基本机制。由于其短的一代人的时间和特点是基因组，实验酵母模型系统的一个主要优势是能够进行遗传筛选，以确定基因和途径，是在一个给定的过程中涉及的。在过去的三十多年，这种遗传屏幕已被用来阐明细胞周期，分泌途径，以及更高度保守的许多方面的真核细胞生物学1-5。在过去的几年里中，几个酵母菌株和质粒基因组库已产生 6-10 。这些藏品现在允许对基因功能的系统，使用增益和损失功能的方法11-16的审讯。在这里，我们提供了一个详细的协议，与液体处理机器人执行质粒的过度表达屏幕使用一种高通量的酵母转化协议使用5500酵母质粒摆着库。我们一直在使用这些画面，找出容易发生聚集人类神经退行性疾病的蛋白质的积累相关的遗传毒性修饰符。这里介绍的方法很容易适应其他感兴趣的细胞表型的研究。

Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

High-throughput Yeast Plasmid Overexpression Screen

在这里，我们描述了一个质粒的过度表达屏幕<em&gt;酿酒酵母</em&gt;，使用液体处理机器人摆着质粒库和高通量酵母转化协议。

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Research

JoVE Journal

Biology

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

一个Biomining从宏基因组库的纤维素酶的活性高通量筛选

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

科研

生物学

High-throughput Saccharification Assay for Lignocellulosic Materials

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High Throughput Microinjections of Sea Urchin Zygotes

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

High Throughput Danio Rerio Energy Expenditure Assay

High Throughput Danio Rerio Energy Expenditure Assay

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...