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

Bu çalışma, Johns Hopkins ALS Araştırma Packard Merkezi (ADG), NIH Müdürü Yenilikçisi Ödülü 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), Rita Allen Vakfı Scholar Ödülü bir hibe ile desteklenmiştir. ADG Pew Charitable Ortaklıkları tarafından desteklenen Biyomedikal Bilimler Pew Scholar.

1. Maya dönüşüm için hazırlıklar Bu protokol on 96-kuyucuğu için tasarlanmıştır ancak bu doğrultuda yukarı veya aşağı ölçeklendirilebilir. Biz bu protokolü, dönüşüm yuvarlak başına yirmiden fazla 96 kuyucuğu için iyi olmadığını bulduk. Tüm dönüşüm prosedürü (adım I.3) yaklaşık sekiz saat sürer. <ol><li> Kısım yuvarlak bir alt her kuyuya Biorobot RapidPlate sıvı işleyici 96-plaka Maya FLEXGene ORF kütüphane plazmid DNA (100 ng / ul) 5-10μL....

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Burada maya yüksek verimli bir plazmid aşırı ekspresyonu ekran gerçekleştirmek için bir protokol mevcut. Bu yaklaşım, birçok farklı hücre fenotipleri genetik nitelemeler için hızlı ve tarafsız bir tarama sağlar. Bu yaklaşımı kullanarak, bir araştırmacı, birkaç hafta içinde maya genomu önemli bir bölümünü ekrana. Bu tarafsız bir yaklaşım aynı zamanda, önceki bulgulara göre tahmin edilmiş olabilir değiştirici belirlenmesi için olanak sağlar. Biz insan nörodejeneratif hastalık pro...

<table border="1"><tbody><tr><th> Reaktif Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 cıvata çoğaltıcı (Frogger) </td><td> V &amp; P Bilimsel </td><td> VP404 </td></tr><tr><td> FLEXGene ORF Kütüphanesi </td><td> Proteomik Enstitüsü, Harvard Tıp Fakültesi </td><td></td></tr><tr><td> Masa üstü santrifüj </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500ml şaşkın şişesi </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L üçlü şaşkın FERNBACH şişesi </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100μL Rapidplate pipet uçları </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200μL Rapidplate pipet uçları </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

Saccharomyces cerevisiae, tomurcuklanan bir maya, insan hastalığı ile doğrudan ilgili olanlar da dahil olmak üzere birçok önemli hücresel süreçlerin temel mekanizmaları, tanımlamak için güçlü bir model sistem. Çünkü, kısa üretim süresi ve iyi karakterize genom, maya model sistem büyük bir deneysel avantajı, belirli bir süreç içinde yer genler ve yolları belirlemek için genetik ekranlar gerçekleştirmek için yeteneğidir. Son otuz yıl içinde bu tür genetik ekranlar hücre döngüsü, salgı yolağı ve ökaryotik hücre biyolojisi 1-5 çok daha yüksek oranda korunmuş yönlerini aydınlatmak için kullanılmaktadır . Son birkaç yıl içinde, maya suşları ve plazmid birkaç genomewide kütüphaneler 6-10 oluşturuldu. Bu koleksiyon, kazanç ve kayıp fonksiyon-yaklaşımları kullanarak 11-16 genin fonksiyonu ile sistematik bir sorgulama için izin verir. Burada dizilmiş 5.500 maya plazmid bir kütüphaneyi kullanarak, plazmid aşırı ekspresyonu ekranı gerçekleştirmek için bir sıvı taşıma robotu ile yüksek verimli bir maya dönüşüm protokolü kullanımı için ayrıntılı bir protokol sağlar. Biz kümeleme eğilimli insan nörodejeneratif hastalık proteinlerin birikimi ile ilişkili toksisite genetik nitelemeler tanımlamak için bu ekranlar kullanıyoruz. Burada sunulan yöntemler diğer ilgi hücre fenotipleri çalışma kolayca uyarlanabilir.

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

High-throughput Yeast Plasmid Overexpression Screen

Burada bir plazmid aşırı ekspresyonu ekran tarif<em&gt; Saccharomyces cerevisiae</em&gt;, Bir sıvı taşıma robotu ile dizilmiş plazmid bir kütüphane ve yüksek verimli bir maya dönüşüm protokolü kullanarak.

Yüksek verim Maya Plazmid overekspresyonu Ekran

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

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