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

Detta arbete stöddes av ett bidrag från Packard Centrum för ALS-forskning vid Johns Hopkins (ADG), en NIH direktörens Nya Innovator Award 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), den Rita Allen Foundation Scholar Award. ADG är en Pew Scholar i Biomedicinsk vetenskap, som stöds av The Pew Charitable Trusts.

1. Förberedelser för jäst omvandling Detta protokoll är avsedd för tio 96-brunnar, men kan skalas upp eller ner därefter. Vi har funnit att detta protokoll inte fungerar bra för mer än tjugo plattor med 96 brunnar per runda av transformation. Hela förvandlingen förfarandet (från steg I.3) tar cirka åtta timmar. <ol><li> Alikvotera 5-10μL av plasmid-DNA (100 ng / l) från jästen FLEXGene ORF bibliotek i varje brunn i en rundbottnad 96-brunnar med Biorobot RapidPlate vätska ha...

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Här presenterar vi ett protokoll för att utföra en hög genomströmning plasmid överuttryck skärm i jäst. Detta tillvägagångssätt möjliggör snabb och objektiva screening för genetiska modifierare av många olika cellulära fenotyper. Med denna metod kan en forskare visa en betydande del av jästgenomet inom några veckor. Detta objektiva tillvägagångssätt gör det möjligt även för identifiering av modifierare, vilket inte kan ha förutsett på grundval av tidigare fynd. Vi har använt denna metod för ...

<table border="1"><tbody><tr><th> Namn på reagens </th><th> Företag </th><th> Katalognummer </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 bult Replicator (Frogger) </td><td> V &amp; P Vetenskapliga </td><td> VP404 </td></tr><tr><td> FLEXGene ORF Bibliotek </td><td> Institutet för Proteomics, Harvard Medical School </td><td></td></tr><tr><td> Tabletop centrifug </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500ml förbryllad kolv </td><td> Bellco </td><td> 2543-00.500 </td></tr><tr><td> 2.8L trippel-förbryllad Fernbach kolv </td><td> Bellco </td><td> 2551-02.800 </td></tr><tr><td> 100μL Rapidplate pipettspetsar </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200μL Rapidplate pipettspetsar </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

Den spirande jäst, Saccharomyces cerevisiae, är en kraftfull modell för att definiera grundläggande mekanismer i många viktiga cellulära processer, inklusive de med direkt relevans för mänskliga sjukdomar. På grund av dess korta generationstid och väldefinierad genomet, är en stor experimentell fördel med jäst modellsystem förmågan att utföra genetiska skärmar för att identifiera gener och signalvägar som är involverade i en viss process. Under de senaste trettio åren sådana genetiska skärmar har använts för att belysa cellcykeln, Utsöndringsvägarna, och många fler starkt konservativa delar av eukaryota cellbiologiska 1-5. Under de senaste åren har flera genomewide bibliotek av jäststammar och plasmider genererats 6-10. Dessa samlingar kan nu för systematisk förhör av geners funktion med hjälp av vinst-och förlust-av-funktionen metoder 11-16. Här ger vi ett detaljerat protokoll för användning av en hög genomströmning jäst omvandling protokoll med ett vätskehantering robot att utföra en plasmid överuttryck skärmen med en klädd bibliotek med 5500 jäst plasmider. Vi har använt dessa skärmar att identifiera genetiska modifierare av toxicitet i samband med ackumulering av aggregering som ofta drabbas människor neurodegenerativa sjukdomar proteiner. Metoderna som presenteras här är lätt att anpassa till studiet av andra cellulära fenotyper av intresse.

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

High-throughput Yeast Plasmid Overexpression Screen

Här beskriver vi en plasmid överuttryck skärm<em&gt; Saccharomyces cerevisiae</em&gt;, Med hjälp av en klädd plasmid bibliotek och en hög genomströmning jäst omvandling protokoll med ett vätskehantering robot.

Med hög kapacitet Jäst Plasmid Överuttryck skärm

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

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