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

Diese Arbeit wurde durch einen Zuschuss aus dem Packard Zentrum für ALS-Forschung an der Johns Hopkins (ADG), ein NIH Director des New Innovator Award 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), der Rita Allen Foundation Scholar Award unterstützt. ADG ist ein Pew Scholar in der Biomedizin, von The Pew Charitable Trusts unterstützt.

1. Die Vorbereitungen für die Hefe-Transformation Dieses Protokoll wird für zehn 96-Well-Platten ausgelegt, kann jedoch vergrößert oder verkleinert entsprechend. Wir haben festgestellt, dass dieses Protokoll nicht gut funktioniert seit mehr als zwanzig 96-Well-Platten pro Runde der Transformation. Die gesamte Transformation Verfahren (aus Schritt I.3) dauert ca. 8 Stunden. <ol><li> Aliquot 5-10 &amp;mgr; l von Plasmid-DNA (100 ng / ul) aus der Hefe FLEXGene ORF-Bibliothek in jede Verti...

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Hier präsentieren wir ein Protokoll zu einem High-Throughput-Plasmid-Überexpression in der Hefe Bildschirm ausführen. Dieser Ansatz ermöglicht die schnelle und unvoreingenommene Screening auf genetische Modifikatoren von vielen verschiedenen zellulären Phänotypen. Mit diesem Ansatz kann ein Forscher einen bedeutenden Teil des Hefe-Genoms in einer Angelegenheit von Wochen-Bildschirm. Diese unvoreingenommene Herangehensweise ermöglicht auch die Identifizierung von Modifikatoren, die nicht vorhergesagt basierend auf...

<table border="1"><tbody><tr><th> Name des Reagenz </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 Bolzen Replikator (frogger) </td><td> V &amp; P Scientific </td><td> VP404 </td></tr><tr><td> FLEXGene ORF-Bibliothek </td><td> Institute of Proteomics, Harvard Medical School </td><td></td></tr><tr><td> Tischzentrifuge </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500mL Schikanekolben </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L Triple-Rätsel Fernbach-Kolben </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100 &amp;mgr; Rapidplate Pipettenspitzen </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200 ul Rapidplate Pipettenspitzen </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

Die Bierhefe, Saccharomyces cerevisiae, ist ein leistungsfähiges Modell für die Definition grundlegenden Mechanismen von vielen wichtigen zellulären Prozessen, einschließlich solcher mit unmittelbarer Relevanz für menschliche Krankheiten. Wegen seiner kurzen Generationszeit und gut charakterisierten Genoms, ist ein großer experimenteller Vorteil der Hefe Modellsystem die Fähigkeit, genetische Screens durchgeführt, um Gene und Signalwege, die in einem bestimmten Prozess beteiligt sind zu identifizieren. Im Laufe der letzten 30 Jahre solche genetischen Screens wurden verwendet, um den Zellzyklus, Sekretionsweg, und viele weitere hochkonservierte Aspekte der eukaryotischen Zelle Biologie 1-5 aufzuklären. In den letzten Jahren haben mehrere genomweiten Bibliotheken Hefestämme und Plasmide wurden 6-10 generiert. Diese Sammlungen erlauben nun für die systematische Befragung der Genfunktion mit Gewinn-und Verlust-of-function Ansätze 11-16. Hier bieten wir Ihnen ein detailliertes Protokoll für den Einsatz eines High-Throughput-Hefe-Transformation-Protokoll mit einem Liquid-Handling-Roboter ein Plasmid Überexpression Bildschirm durchführen, mit einem Array-Bibliothek von 5.500 Hefeplasmide. Wir verwenden diese Bildschirme zu genetischen Modifikatoren der Toxizität mit der Akkumulation von Aggregation neigende menschliche neurodegenerative Erkrankung Proteinen assoziiert zu identifizieren. Die hier vorgestellten Methoden sind leicht an das Studium der anderen zellulären Phänotypen von Interesse.

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

High-throughput Yeast Plasmid Overexpression Screen

Hier beschreiben wir ein Plasmid Überexpression Bildschirm in<em&gt; Saccharomyces cerevisiae</em&gt;, Mit einem Array-Plasmid-Bibliothek und einem High-Throughput-Hefe-Transformation-Protokoll mit einem Liquid-Handling-Roboter.

High-throughput Hefeplasmid Überexpression Bildschirm

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

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