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

Este trabalho foi financiado por uma doação do Centro de Packard de Investigação em ALS Johns Hopkins (ADG), um diretor Award NIH Innovator New 1DP2OD004417-01 (ADG), R01 NIH NS065317 (ADG), a Rita Allen Foundation Scholar Award. ADG é um Scholar Pew em Ciências Biomédicas, apoiado pelo Pew Charitable Trusts.

1. Preparações para a transformação de levedura Este protocolo é projetado para dez placas de 96 poços, mas podem ser ampliados ou reduzidos em conformidade. Descobrimos que este protocolo não funciona bem por mais de vinte placas de 96 poços por rodada de transformação. O procedimento de transformação inteira (a partir do passo I.3) levará cerca de oito horas. <ol><li> Alíquota de 5 10μL de DNA plasmídeo (100 ng / mL) a partir da levedura biblioteca ORF FLEXGene em todos o...

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Aqui apresentamos um protocolo para realizar um alto throughput tela superexpressão plasmídeo em levedura. Esta abordagem permite a detecção rápida e imparcial de modificadores genéticos de diferentes fenótipos celulares. Usando essa abordagem, um pesquisador pode selecionar uma porção significativa do genoma da levedura em questão de semanas. Esta abordagem imparcial também permite a identificação de modificadores, que podem não ter sido previsto com base em resultados anteriores. Nós temos usado essa ab...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 parafuso replicador (frogger) </td><td> V &amp; P Scientific </td><td> VP404 </td></tr><tr><td> FLEXGene ORF Biblioteca </td><td> Instituto de Proteomics, Harvard Medical School </td><td></td></tr><tr><td> Centrífuga de mesa </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> Frasco perplexo 500mL </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L triple-perplexo frasco Fernbach </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> Rapidplate 100μL ponteiras </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> Rapidplate 200μL ponteiras </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

A levedura de brotamento, Saccharomyces cerevisiae, é um sistema poderoso modelo para a definição de mecanismos fundamentais de muitos processos celulares importantes, incluindo aquelas com relevância direta para a doença humana. Por causa de seu tempo de geração curto e bem caracterizadas do genoma, uma grande vantagem experimental do sistema de modelo de levedura é a capacidade de realizar triagem genética para identificar genes e caminhos que estão envolvidos em um determinado processo. Nos últimos 30 anos como telas genética têm sido usadas para elucidar o ciclo celular, via de excreção, e muitos outros aspectos altamente conservadas da biologia das células eucarióticas 1-5. No últimos anos, diversas bibliotecas do genoma de cepas de leveduras e plasmídeos foram gerados 6-10. Estas colecções permitem agora para o interrogatório sistemático da função do gene usando-ganho e perda de função abordagens 11-16. Aqui nós fornecemos um protocolo detalhado para o uso de um protocolo de transformação de alto rendimento fermento com um robô para manipulação de líquidos para executar uma tela superexpressão plasmídeo, usando uma biblioteca de vestidos de 5.500 plasmídeos de levedura. Temos vindo a utilizar estas telas para identificar modificadores genéticos de toxicidade associado ao acúmulo de agregação propensas proteínas humanas doença neurodegenerativa. Os métodos aqui apresentados são facilmente adaptáveis ​​para o estudo de outros fenótipos celulares de interesse.

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

High-throughput Yeast Plasmid Overexpression Screen

Aqui nós descrevemos uma tela superexpressão plasmídeo em<em&gt; Saccharomyces cerevisiae</em&gt;, Usando uma biblioteca vestiu plasmídeo e uma alta taxa de transferência de protocolo de transformação de levedura com um robô para manipulação de líquidos.

Alto rendimento Yeast Tela superexpressão Plasmid

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

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