|
| Status |
Public on Feb 28, 2007 |
| Title |
ADH1 deletion mutant grown in glycerol (2 hour) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ADH1 deletion mutant grown in glycerol (2 hour)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain: BY4741 ADH1::KANMX, haploid
|
| Growth protocol |
adh1 cells were grown overnight on YPD (2% glucose) to O.D. 0.4, washed and then diluted into either YP-glycerol (2%) or fresh YPD
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected, pelleted and immediately frozen.Total RNA was obtained using MasterPure yeast RNA purification kit (Epicenter).
|
| Label |
Cy5
|
| Label protocol |
cDNA was synthesized from total RNA using M-MLV Reverse TranscriptaseRNase H Minus (Promega) and labeled by the indirect amino-allyl method.
|
| |
|
| Channel 2 |
| Source name |
ADH1 deletion mutant grown overnight on YPD (glucose)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain: BY4741 ADH1::KANMX, haploid
|
| Growth protocol |
adh1 cells were grown overnight on YPD to O.D. 0.4
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected, pelleted and immediately frozen.Total RNA was obtained using MasterPure yeast RNA purification kit (Epicenter).
|
| Label |
Cy3
|
| Label protocol |
cDNA was synthesized from total RNA using M-MLV Reverse TranscriptaseRNase H Minus (Promega) and labeled by the indirect amino-allyl method.
|
| |
|
| |
| Hybridization protocol |
cDNA samples were labeled with Cy3 and Cy5 and combined with blockers: 5?g Herring sperm (Promega), 5?g tRNA (Gibco) and 17.5?g Poly A (Poly A oligos were synthesized with mixed length of 40, 50 and 60 Adenine residues). The labeled cDNAs were concentrated to 40?l using Microcon (Millipore) and 40?l of hybridization x2 solution (10x SSC, 50% formamide and 0.2% SDS) was added. Microarrays containing all yeast ORFs were pre-hybridized by incubating at 42C for 45 minutes in a solution containing 1% BSA, 25% formamide, 5x SSC and 0.1% SDS. The slides were washed in sterile water and dried by centrifugation (3 minutes, 2000 rpm). The labeled samples were boiled for 5 minutes, centrifuged for 1 minute, hybridized on the slide and placed in a hybridization chamber (Corning) for overnight incubation at 42C. The slides were then washed for 5 minutes at 42C with a solution containing 2x SSC and 0.1% SDS. Additional wash was performed at room temperature with a solution containing 0.1x SSC and 0.1% SDS, followed by three additional washes at room temperature in 0.1x SSC solution.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 using Aginlet DNA microarray scannerand image intensity data were extracted and analyzed with SpotReader (Niles Scientific) analysis software.
|
| Description |
ADH1 deletion mutant grown in glycerol (2 hour)
|
| Data processing |
Background intensity was removed by subtracting the mean array intensity and the ratios of the two dyes were log2-transformed. The two spots corresponding to each gene were then averaged, and genes with low intesity were excluded by manual inspection).
|
| |
|
| Submission date |
Nov 16, 2006 |
| Last update date |
Feb 28, 2007 |
| Contact name |
sagi levy |
| E-mail(s) |
sagi.levy@weizmann.ac.il
|
| Phone |
972-8-9342201
|
| URL |
http://barkai-serv.weizmann.ac.il/GroupPage/
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Molecular Genetics and Physics of Complex Systems
|
| Lab |
Naama Barkai
|
| Street address |
Hertzel
|
| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL4567 |
| Series (1) |
| GSE6302 |
ADH1 deletion cells upon growth on alternative carbon sources; Steady-state grown cells upon environmental perturbations |
|
