|
| Status |
Public on Jun 21, 2008 |
| Title |
CDC34tm strain, replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
total RNA, experimental variables include CDC34tm mutation, a nourseothricin resistance gene, partial disruption of PST1 3' region and leu2-3,112 allele
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
CDC34tm confers only slightly slow growth phenotype
|
| Treatment protocol |
Cells frozen in liquid nitrogen
|
| Growth protocol |
The Saccharomyces cerevisiae CDC34tm strain (RRC85, MATα, CDC34tm(NatR)) which is marked by a nourseothricin resistance gene (NatR) and an isogenic wild type strain (DBY2059, MATα, leu2-3, 112) were inoculated from stationary phase cultures into a synthetic defined medium containing 2% dextrose, 0.17% yeast nitrogen base minus amino acids and ammonium sulfate, 0.25% L-glutamine, 0.025% magnesium sulfate and 25.2 mg/L L-Leucine. Four separate cultures of each strain were grown at 30°C, allowing between three and four doublings and cells were collected at approximately 8 x 10e6cells/ml.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a hot acid phenol-chloroform protocol as previously described (Schmitt et al. 1990). RNA quality was verified with OD260/280 readings and a 1.5% agarose gel.
|
| Label |
biotin
|
| Label protocol |
For samples of 2 microgram or more, we do single cycle labeling. This involves synthesizing cDNA using a T7 promoter-dT24 oligonucleotide as primer with the Invitrogen Life Technologies SuperScript Choice system. Following second strand cDNA synthesis and incubation with T4 DNA polymerase, the products are purified using an Affymetrix Cleanup Module. Biotinylated cRNA is made using the Affymetrix IVT kit.
|
| |
|
| Hybridization protocol |
The cRNA is purified using Qiagen RNeasy columns, quantitated and then fragmented by incubation at high temperature with magnesium. Fifteen μg of biotinylated cRNA is added to a total hybridization cocktail of 300 μl, and 200 μl is hybridized to a GeneChip® after adding control oligonucleotides. Hybridization is at 45°C for 17 h with constant rotation. The hybridization mixture is then removed and the GeneChips® are washed, stained with phycoerythrin-labeled Streptavidin, washed, incubated with biotinylated anti-streptavidin, and then restained with phycoerythrin-labeled Streptavidin to amplify the signals; these steps are carried out using the Affymetrix Fluidics Station. To reduce non-random error, balanced groups of samples are handled in parallel.
|
| Scan protocol |
Arrays are then scanned using the dedicated scanner, controlled by Affymetrix GCOS software. Images are examined for defects.
|
| Description |
no additional
|
| Data processing |
The Affymetrix® Microarray Suite version 5 (MAS5) algorithm analyzes the hybridization intensity data from GeneChip® expression probe arrays and calculates a set of metrics that describe probe set performance. The average intensity on each array is normalized by global scaling to a target intensity of 1000.
|
| |
|
| Submission date |
Jul 12, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Mark Goebl |
| E-mail(s) |
mgoebl@iupui.edu
|
| Phone |
317-274-2055
|
| Fax |
317-278-9739
|
| URL |
http://www.biochemistry.iu.edu/personnel/Goebl/goebllab/goebl.htm
|
| Organization name |
Indiana University School of Medicine
|
| Department |
Biochemistry and Molecular Biology
|
| Lab |
Mark Goebl
|
| Street address |
1345 West 16th St.
|
| City |
Indianapolis |
| State/province |
IN |
| ZIP/Postal code |
46202 |
| Country |
USA |
| |
|
| Platform ID |
GPL2529 |
| Series (1) |
| GSE8453 |
Expression data from yeast strain containing CDC34tm allele compared to WT |
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