|
| Status |
Public on Nov 17, 2007 |
| Title |
BQS252_thiolutin_3ug t30min replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
yeast strain FY1679 derived growing in exponential phase treated with 3 µ/mL thiolutin t30min
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Total RNA from BQS252 yeast strain (Mat a, ura3-52 derived from FY1679) growing in exponential phase in YPD
|
| Biomaterial provider |
total RNA
|
| Treatment protocol |
30 minutes after the addition of thiolutin 3 µ/mL
|
| Growth protocol |
Total RNA from BQS252 yeast strain growing in exponential phase in YPD (yeast extract 1%; peptone 2%,glucose 2%)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from yeast cells was prepared as described by
Sherman et al. [1986], but using a multiple-sample automated
device (Fast-Prep, BIO101) to break the cells.
Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics.
Spring Harbor Cold Laboratory Press, Cold Spring Harbor, New York
|
| Label |
33PdCTP
|
| Label protocol |
About 30-40 µg of total RNA were reverse transcribed into cDNA by adding 200 us of RT polymerase SuperScript II and random hexamers (Invitrogen), 1 µL RNaseOUT (Invitrogen), 6 µL 5X First Strand Buffer (Invitrogen), 1.5 µL dNTP mix (16 mM dATP, dTTP, dGTP, and 100 µM dCTP) and 5 µL [α-33P]dCTP (3000 Ci/mmol, 10 µCi/microL) in a final reaction volume of 30 µL. The labeling reaction was incubated for 1 h at 43ºC. 1 µL of EDTA 0.5 M was added to stop the reaction. The labeled cDNAs were purified by a S300-HR MicroSpin column (Amersham BioSciences).
|
| |
|
| Hybridization protocol |
Hybridization Solution was: 5XSSC, 5XDenhart’s, 0.5% SDS and 100 µg herring sperm DNA/ml.
The hybridization protocol used was as follows. Filters were inserted in 12.5X2.5-cm flat-bottom plastic tubes and pre-hybridized in a rotator oven with 5 ml pre-hybridization solution (the same as used for hybridization but without the radioactive sample) at 65ºC. The pre-hybridization solution was then replaced with 5 ml of the same solution containing 3X10exp6 dpm/mL of radioactive sample and hybridized for 44h. Washing conditions were: 1h at 65ºC for 20 min in 2XSSC, 0.1% SDS, and twice at 65ºC for 30 min in 0.2XSSC, 0.1% SDS.
|
| Scan protocol |
Images were acquired using a FujiFilm FLA3000 Phosphorimager.
|
| Description |
Filters were stripped by pouring 3X150 ml boiling stripping buffer (5 mM sodium phosphate, pH 7.5, 0.1% SDS) over the
membrane. The first time, the stripping buffer was immediately changed while after the second and third washes the filters
were left to cool at room temperature. To ensure that radioactivity had been eliminated, the filters were either checked
with a Geiger counter or re-scanned with a Phosphorimager.
|
| Data processing |
Raw image quantization background subtracted
|
| |
|
| Submission date |
Mar 14, 2007 |
| Last update date |
Nov 26, 2007 |
| Contact name |
Jose E. Perez-Ortin |
| E-mail(s) |
jose.e.perez@uv.es
|
| Phone |
34 963 543467
|
| Organization name |
Universitat de Valencia
|
| Department |
Bioquimica y Biologia Molecular
|
| Lab |
Yeast Functional Genomics (GFL)
|
| Street address |
Dr. Moliner 50
|
| City |
Burjassot |
| State/province |
Valencia |
| ZIP/Postal code |
E46100 |
| Country |
Spain |
| |
|
| Platform ID |
GPL3763 |
| Series (2) |
| GSE7261 |
Yeast mRNA decay analysis after addition of 3 µ/mL thiolutin |
| GSE8629 |
Yeast mRNA decay analysis after addition of 3 & 10 µ/mL thiolutin |
|
