Summary
このプロトコルは、酵母の遺伝子発現（内
Abstract
このプロトコルでは、酵母の遺伝子発現（ 出芽酵母 ） は、過酸化水素（H 2 O 2）、酸化剤の添加により誘導される酸化ストレスに暴露した後に変更されます。実験では、酵母は3Xグルコースを含む2分の1にYPD液体培地で48時間増殖されています。培養は、対照と投与群に分割されます。実験の文化を0.5mmハンクスのH 2 O 2で処理されること1時間（HBSS）緩衝生理食塩水。対照培養は、HBSSで扱われます。トータルRNAは、両方の文化から抽出され、多段階のプロセスを介してビオチン標識cRNAの製品に変換されます。最終的な合成製品は、UVMマイクロアレイの中核施設に戻って採取し、アフィメトリクスの酵母GeneChipsにハイブリダイズさせる。得られた遺伝子発現データは、バイオインフォマティクスデータ解析ソフトウェアにアップロードされます。
Protocol
Discussion
Disclosures
このビデオ記事の生産は、EMD -ミリポアが後援した。
Acknowledgments
Materials
| Name | Company | Catalog Number | Comments |
| Spetrophotometer | |||
| Thermocyler | |||
| Microcentrifuge | |||
| Vortex | |||
| Stir Plates (2) | |||
| P-10s pipettors | |||
| P-20's pipettors: | |||
| P-200's pipettors: | |||
| P-1000's pipettors: | |||
| Camera and transilluminator | |||
| E-Gel Apparatus | Invitrogen | G6000-08 | |
| E-Gels | Invitrogen | G6018-02 | |
| Axygen 1.7 ml. Clear | Krackeler Scientific, Inc. | 383-MCT175C | |
| Axygen 0.5 ml clear tubes | Krackeler Scientific, Inc. | 383-MCT060C | |
| Axygen 2ml capture tubes | Krackeler Scientific, Inc. | 383-MCT200NC | |
| boxes of P-10 ART tips: | CLP | BT10XL | |
| Boxes of P-100 ART tips: | CLP | BT200 | |
| Boxes of P-20 ART Tips: | CLP | BT20 | |
| Boxes of P-1000 ART Tips: | CLP | BT1000 | |
| Yeast Chips | |||
| 25 ml sterile disposable pipets- | |||
| 5ml sterile disposable pipets- | |||
| Microfuge tube racks | |||
| KimWipes: | |||
| Lab Coats | |||
| Stir Bars | |||
| Culture flasks | |||
| Innoculating loops | |||
| Sharpies | |||
| Gloves | |||
| Autoclave tape | |||
| Stirrer bars | |||
| 30% Hydrogen Peroxide |  EMD Millipore |
386790 | |
| HBSS without Ca, Mg, or dye | Sigma-Aldrich | H6648-100ml | |
| Qiagen Rneasy Mini Kit: | Qiagen | 74104 | |
| Qiagen DNase Kit: | Qiagen | 79254 | |
| Rnase Remover | Denville Scientific | D1180 | |
| Harleco Alcohol 100% | EMD Millipore |
65347 | |
| ENZO IVT Kit | Enzo Life Sciences | ENZ-42655-10 | |
| Lyticase: one bottle | VWR international | IC19012310 | |
| Water, Cell Culture grade | EMD Millipore |
4.86505 | |
| Yeast culture: | ATCC | 18824 | |
| YPD broth powder | EMD Millipore |
4.8504 | |
| D(+) Glucose, Anhydrous | EMD Millipore |
346351 | |
| Sorbitol | EMD Millipore |
56755 | |
| EDTA | EMD Millipore |
4005 | |
| SDS solution, 20% | EMD Millipore |
7990-OP | |
| Pellet Paint | EMD Millipore |
69049 | |
| PCI RNase DNase free (7 aliquots): | Fisher Scientific | AC32711-500 | |
| 7.5M NH4OAc | Fisher Scientific | ICN1987 5980 | |
| Fragmentation Buffer (200 mM Tris-Acetate, pH 8.1, 500 mM KOAc, 150 mM MgOA) | |||
| Trizma Base | Fiaher | 50-899-90034 | |
| KOAc | Fisher Scientific | NC9757725 | |
| MgOA | Fisher Scientific | NC9681042 | |
| Loading Dye | Invitrogen | 10482055 | |
| PCR Markers | EMD Millipore |
69278-3 | |
| Phase Lock Gels | Fisher Scientific | FP2302820 | |
| One-Cycle cDNA Kit | Invitrogen | A10752-030 | |
| Includes; | |||
| E. coli DNA Pol | |||
| dNTP's | |||
| T4 DNA Pol. | |||
| T-7 oligod(T) | |||
| 0.5ml EDTA pH 8.0 | |||
| First Strand Buffer | |||
| E. Coli RNaseH | |||
| Second Strand Buffer | |||
| E. Coli DNA ligase | |||
| SuperScript II | |||
| DTT |
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