Summary
In diesem Protokoll, Genexpression in Hefe (
Abstract
In diesem Protokoll wird die Genexpression in Hefe (Saccharomyces cerevisiae) verändert nach Exposition mit oxidativem Stress durch die Zugabe von Wasserstoffperoxid (H 2 O 2), ein Oxidationsmittel induziert. Im Experiment wird Hefe für 48 Stunden in 1/2X YPD Brühe mit 3X Glukose gewachsen. Die Kultur wird in eine Kontroll-und behandelten Gruppe aufgeteilt. Das Experiment Kultur mit 0,5 mM H 2 O 2 in Hanks behandelt Kochsalzlösung (HBSS) für 1 Stunde. Die Kontroll-Kultur mit HBSS nur behandelt. Gesamt-RNA wird aus beiden Kulturen extrahiert und mit einem Biotin-markierten cRNA Produkt durch ein mehrstufiger Prozess umgewandelt. Die endgültige Synthese Produkt wieder auf die UVM Microarray Core Facility genommen und hybridisiert, die Affymetrix Hefe GeneChips. Die daraus resultierende Genexpression Daten werden in der Bioinformatik Datenanalyse-Software hochgeladen.
Protocol
Discussion
Disclosures
Die Produktion dieses Video-Artikel wurde von EMD-Millipore gesponsert.
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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