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

Ce travail a été soutenu par une subvention du Centre pour la recherche sur la SLA Packard à la Johns Hopkins (ADG), Prix d&#39;un directeur des NIH Innovateur Nouveau 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), la Fondation Rita Allen Scholar Award. ADG est une bourse d&#39;études Pew dans les sciences biomédicales, soutenu par le Pew Charitable Trusts.

1. Les préparatifs pour la transformation de la levure Ce protocole est conçu pour les dix plaques de 96 puits, mais peut être agrandie ou réduite en conséquence. Nous avons trouvé que ce protocole ne fonctionne pas bien depuis plus de vingt plaques de 96 puits par tour de transformation. La procédure de transformation complète (de l&#39;étape I.3) prendra environ huit heures. <ol><li> Aliquote de 5-10 ul d&#39;ADN plasmidique (100 ng / uL) de la bibliothèque de levure FLEXGene O...

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Nous présentons ici un protocole pour effectuer un écran haut débit surexpression plasmide dans la levure. Cette approche permet le criblage rapide et impartiale pour les modificateurs génétiques de beaucoup de différents phénotypes cellulaires. En utilisant cette approche, un chercheur peut filtrer une partie importante du génome de la levure dans une affaire de semaines. Cette approche impartiale permet également d&#39;identifier les modificateurs, qui peuvent ne pas avoir été prédit sur la base des conclu...

<table border="1"><tbody><tr><th> Nom de réactif </th><th> Société </th><th> Le numéro de catalogue </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 boulons réplicateur (Frogger) </td><td> V &amp; P scientifique </td><td> VP404 </td></tr><tr><td> FLEXGene ORF Bibliothèque </td><td> Institut de la protéomique, la Harvard Medical School </td><td></td></tr><tr><td> Centrifugeuse de table </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500 ml flacon dérouté </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L triple dérouté flacon Fernbach </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100 ul Rapidplate pipette </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200 pl Rapidplate pipette </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

Le bourgeonnement de levure Saccharomyces cerevisiae, est un système puissant modèle pour définir les mécanismes fondamentaux de nombreux processus cellulaires importants, y compris ceux en rapport direct avec la maladie humaine. En raison de son temps de génération court et bien caractérisés génome, un avantage majeur d&#39;expérimentation du système modèle la levure est la capacité à effectuer des criblages génétiques pour identifier les gènes et les voies qui sont impliquées dans un processus donné. Au cours des trente dernières années tels cribles génétiques ont été utilisés pour élucider le cycle cellulaire, voie de sécrétion, et de nombreux autres aspects de la biologie hautement conservée cellule eucaryote 1-5. Au cours des dernières années, plusieurs bibliothèques genomewide des souches de levures et plasmides ont été générés 6-10. Ces collections permettent maintenant de l&#39;interrogation systématique de la fonction des gènes en utilisant gain et la perte de fonction des approches 11-16. Ici nous fournissons un protocole détaillé pour l&#39;utilisation d&#39;un protocole de haut débit de la levure de transformation avec un robot de manipulation de liquides pour effectuer un écran surexpression plasmidique, en utilisant une bibliothèque de 5.500 vêtu plasmides de levure. Nous avons utilisé ces écrans pour identifier modificateurs génétiques de la toxicité associée à l&#39;accumulation de l&#39;agrégation des protéines humaines exposées aux maladies neurodégénératives. Les méthodes présentées ici sont facilement adaptables à l&#39;étude d&#39;autres phénotypes cellulaires d&#39;intérêt.

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

High-throughput Yeast Plasmid Overexpression Screen

Nous décrivons ici un écran surexpression plasmide dans<em&gt; Saccharomyces cerevisiae</em&gt;, En utilisant une bibliothèque vêtu plasmide et un protocole de haut débit de la levure de transformation avec un robot de manipulation de liquides.

Haut débit écran surexpression plasmide de levure

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