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Microarray-Analyse für Saccharomyces cerevisiae

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Vermont Genetics Network, The University of Vermont

 

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Cite this Article: Microarray-Analyse für Saccharomyces cerevisiae

Tighe, S., Hunter, T., Reed, P., Murray, J. Microarray Analysis for Saccharomyces cerevisiae. J. Vis. Exp. (50), e2585, doi:10.3791/2585 (2011).

Abstract: Microarray-Analyse für Saccharomyces cerevisiae

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: Microarray-Analyse für Saccharomyces cerevisiae

Discussion: Microarray-Analyse für Saccharomyces cerevisiae

Disclosures: Microarray-Analyse für Saccharomyces cerevisiae

Die Produktion dieses Video-Artikel wurde von EMD-Millipore gesponsert.

Acknowledgements: Microarray-Analyse für Saccharomyces cerevisiae

Materials: Microarray-Analyse für Saccharomyces cerevisiae

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

References: Microarray-Analyse für Saccharomyces cerevisiae

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  6. Vermont Genetics Network. Microarray Outreach. http://www.uvm.edu/~vgn/outreach/microarrayoutreach (2008).
  7. Affymetrix GeneChip Expression Analysis: Technical Manual. 701021 Rev. 5. Santa Clara, CA, Affymetrix; (2004). http://www.affymetrix.com/support/technical/manual/expression_manual.affx
  8. Affymetrix GeneChip Expression Analysis: Data Analysis Fundamentals. 701190 Rev. 4. Santa Clara, CA, Affymetrix; [http:/ / www.affymetrix.com/ Auth/ support/ downloads/ manuals/data_analysis_fundamentals_manual.p df] (2004).
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