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

Questo lavoro è stato sostenuto da una borsa di studio del Centro per la SLA Packard ricerca presso la Johns Hopkins (ADG), Premio New Innovator un direttore NIH 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), Rita Allen Foundation Scholar Award. ADP è uno studioso Pew nel Scienze Biomediche, sostenuto dal Pew Charitable Trusts.

1. I preparativi per la trasformazione del lievito Questo protocollo è progettato per una decina di piastre a 96 pozzetti, ma può essere scalato verso l&#39;alto o verso il basso di conseguenza. Abbiamo scoperto che questo protocollo non funziona bene per più di venti piastre a 96 pozzetti per ogni turno di trasformazione. La procedura di trasformazione intera (dal punto I.3) dura circa otto ore. <ol><li> Aliquota 5-10μL di DNA plasmidico (100 ng / mL) dalla libreria lievito FLEXGene O...

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Qui vi presentiamo un protocollo per eseguire un elevato throughput schermo plasmide sovraespressione nel lievito. Questo approccio permette di screening rapido e imparziale per modificatori genetici di molti diversi fenotipi cellulari. Usando questo approccio, un ricercatore può schermo una porzione significativa del genoma del lievito in una questione di settimane. Questo approccio imparziale consente inoltre l&#39;identificazione di modifica, che non possono essere previste in base ai risultati precedenti. Abbiamo u...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 bullone replicatore (Frogger) </td><td> V &amp; P scientifico </td><td> VP404 </td></tr><tr><td> FLEXGene ORF Biblioteca </td><td> Istituto di Proteomica, Harvard Medical School </td><td></td></tr><tr><td> Centrifuga da tavolo </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500mL fiasco sconcertato </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L triplo sconcertato Fernbach fiasco </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100μL Rapidplate puntali </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200μL Rapidplate puntali </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

Il lievito, Saccharomyces cerevisiae, è un sistema potente modello per la definizione di meccanismi fondamentali di molti importanti processi cellulari, tra cui quelle con rilevanza diretta per la malattia umana. A causa della sua breve tempo di generazione e ben caratterizzati genoma, uno dei principali vantaggi sperimentale del sistema modello lievito è la capacità di eseguire schermi genetico per identificare i geni e le vie che sono coinvolti in un dato processo. Nel corso degli ultimi 30 anni tale schermi genetici sono stati utilizzati per spiegare il ciclo cellulare, via secretoria, e molti altri aspetti altamente conservato della biologia delle cellule eucariotiche 1-5. Negli ultimi anni, diverse biblioteche genoma dei ceppi di lievito e plasmidi sono stati generati 6-10. Queste raccolte permettono ora per l&#39;interrogatorio sistematico della funzione del gene utilizzando guadagno e la perdita di funzione approcci 11-16. Qui forniamo un protocollo dettagliato per l&#39;utilizzo di un high-throughput protocollo di lievito di trasformazione con un robot di gestione dei liquidi per eseguire uno schermo plasmide iperespressione, utilizzando una libreria di 5.500 schierati plasmidi lievito. Abbiamo utilizzato questi schermi per identificare i modificatori genetici di tossicità associata con l&#39;accumulo di proteine ​​umane di aggregazione a rischio di malattie neurodegenerative. I metodi presentati qui sono facilmente adattabili allo studio di altri fenotipi cellulari di interesse.

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

High-throughput Yeast Plasmid Overexpression Screen

Qui si descrive uno schermo plasmide sovraespressione in<em&gt; Saccharomyces cerevisiae</em&gt;, Utilizzando una libreria schierate plasmide e un elevato throughput protocollo di trasformazione di lievito con un robot di manipolazione dei liquidi.

High-throughput iperespressione schermo lievito plasmidi

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