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Microarray analisi per Saccharomyces cerevisiae

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Vermont Genetics Network, The University of Vermont

 

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Cite this Article: Microarray analisi per 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 analisi per Saccharomyces cerevisiae

In questo protocollo, l'espressione genica nel lievito (Saccharomyces cerevisiae) è cambiata dopo l'esposizione a stress ossidativo indotto con l'aggiunta di perossido di idrogeno (H 2 O 2), un agente ossidante. Nell'esperimento, lievito è cresciuto per 48 ore in 1/2X brodo YPD contenenti glucosio 3X. La cultura è diviso in un gruppo di controllo e trattati. La cultura esperimento viene trattata con 0,5 mm H 2 O 2 in Hanks Buffered Saline (HBSS) per 1 ora. La cultura del controllo è trattata con HBSS solo. RNA totale è estratto da entrambe le culture e viene convertito in un biotina marcata cRNA prodotto attraverso un processo a più fasi. Il prodotto finale di sintesi viene riportato al Fondo UVM Nucleo Microarray e ibridato a GeneChip il lievito di Affymetrix. La risultante dati di espressione genica sono stati caricati nel software di analisi bioinformatica dei dati.

Protocol: Microarray analisi per Saccharomyces cerevisiae

Discussion: Microarray analisi per Saccharomyces cerevisiae

Disclosures: Microarray analisi per Saccharomyces cerevisiae

La produzione di questo video-articolo è stato sponsorizzato da EMD-Millipore.

Acknowledgements: Microarray analisi per Saccharomyces cerevisiae

Materials: Microarray analisi per 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 analisi per Saccharomyces cerevisiae

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