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

Dit werk werd ondersteund door een subsidie ​​van het Packard Center voor ALS onderzoek in Johns Hopkins (ADG), Nieuw-Innovator een NIH Director&#39;s Award 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), de Rita Allen Foundation Scholar Award. ADG is een Pew Scholar in de Biomedische Wetenschappen, ondersteund door The Pew Charitable Trusts.

1. De voorbereidingen voor gist transformatie Dit protocol is bedoeld voor tien 96-wells platen, maar kan worden opgeschaald naar boven of beneden aangepast. We hebben ontdekt dat dit protocol niet goed werkt voor meer dan twintig 96-well platen per ronde van transformatie. De gehele transformatie procedure (vanaf stap I.3) duurt ongeveer acht uur. <ol><li> Aliquot 5-10μL van plasmide DNA (100 ng / ul) van de gist FLEXGene ORF bibliotheek in elke well van een ronde bodem 96-well plaat met...

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Hier presenteren wij een protocol om een ​​high-throughput plasmide overexpressie screen in gist uit te voeren. Deze aanpak maakt het mogelijk om snelle en onpartijdige screening op genetische factoren van een groot aantal verschillende cellulaire fenotypes. Met behulp van deze aanpak, een onderzoeker kan een aanzienlijk deel van de gist genoom scherm in een kwestie van weken. Deze onpartijdige aanpak maakt het ook mogelijk voor de identificatie van modifiers, die niet mag worden voorspeld op basis van eerdere bevin...

<table border="1"><tbody><tr><th> Naam van het reagens </th><th> Vennootschap </th><th> Catalogusnummer </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 bout replicator (frogger) </td><td> V &amp; P Scientific </td><td> VP404 </td></tr><tr><td> FLEXGene ORF Bibliotheek </td><td> Instituut van Proteomics, de Harvard Medical School </td><td></td></tr><tr><td> Tafelblad centrifuge </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500ML verbijsterd fles </td><td> Bellco </td><td> 2543 tot 00.500 </td></tr><tr><td> 2.8L triple-verbijsterd Fernbach fles </td><td> Bellco </td><td> 2.551 tot 02800 </td></tr><tr><td> 100μL Rapidplate pipettips </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200μL Rapidplate pipettips </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

De gist Saccharomyces cerevisiae, is een krachtig model systeem voor het definiëren van fundamentele mechanismen van de vele belangrijke cellulaire processen, waaronder die met directe relevantie voor de menselijke ziekte. Vanwege de korte generatietijd en goed gekarakteriseerd genoom, een grote experimentele voordeel van de gist modelsysteem is de mogelijkheid om genetische screens uit te voeren om genen en paden die betrokken zijn bij een bepaald proces te identificeren. In de afgelopen dertig jaar een dergelijke genetische screens zijn gebruikt om de celcyclus, secretieroute, en nog veel meer sterk geconserveerde aspecten van de eukaryote cel biologie 1-5 toe te lichten. In de afgelopen jaren zijn er verschillende genoomwijde bibliotheken van gist stammen en plasmiden is gegenereerd 6-10. Deze collecties nu zorgen voor de systematische ondervraging van gen-functie met behulp van winst-en verlies-van-functie benaderingen 11-16. Hier hebben we een gedetailleerd protocol voor het gebruik van een high-throughput gist transformatie protocol met een liquid handling robot aan een plasmide overexpressie scherm uit te voeren, met behulp van een gekleed bibliotheek van 5.500 gist plasmiden. We hebben gebruik gemaakt van deze schermen aan genetische factoren van toxiciteit in verband met de accumulatie van aggregatie-gevoelige humane neurodegeneratieve ziekten eiwitten te identificeren. De methoden die hier gepresenteerd zijn gemakkelijk aan te passen aan de studie van andere cellulaire fenotypes van belang.

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

High-throughput Yeast Plasmid Overexpression Screen

Hier beschrijven we een plasmide overexpressie scherm in<em&gt; Saccharomyces cerevisiae</em&gt;, Met behulp van een plasmide gekleed bibliotheek en een high-throughput gist transformatie protocol met een liquid handling robot.

High-throughput gist Plasmide Overexpressie Screen

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

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