Summary
I detta protokoll genuttryck i jäst (
Abstract
I detta protokoll är genuttryck i jäst (Saccharomyces cerevisiae) ändras efter exponering för oxidativ stress genom tillsats av väteperoxid (H 2 O 2), ett oxidationsmedel. I experimentet är jäst odlas för 48 timmar i 1/2X YPD buljong innehåller 3X glukos. Kulturen är uppdelat i en kontroll och behandlade gruppen. Experimentet kulturen behandlas med 0,5 mm H 2 O 2 snodda saltlösning (HBSS) i 1 timme. Kontrollen kulturen behandlas med HBSS bara. Total RNA extraheras från båda kulturerna och omvandlas till en biotin-märkt Crna produkt genom en flerstegsprocess. Den slutliga syntesen produkten tas tillbaka till UVM Microarray Core Facility och hybridiseras till Affymetrix jästen GeneChips. Den resulterande genexpression uppgifter laddas upp i bioinformatik dataanalys programvara.
Protocol
Discussion
Disclosures
Produktionen av denna video-artikeln var sponsrad av EMD-Millipore.
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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