|
| Status |
Public on Jul 15, 2008 |
| Title |
genomic V2-L2-F19 |
| Sample type |
genomic |
| |
|
| Source name |
3002 strain
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
V2-L2-F19 filter hybridization with labeled yeast genomic (3002) DNA. DNA labeling by random priming procedure and 33P-dCTP
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from yeast cells by phenol extraction with glass beads, essentially as described by Hoffman CS, Winston F (1987). Ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli (Gene 57:267-272).
|
| Label |
33P-dCTP
|
| Label protocol |
Yeast genomic DNA was labeled following a random priming procedure. 800 ng genomic DNA in 45 µl distilled water was denatured for 5 minutes by boiling, added to a microcentrifuge tube of Ready-To-Go DNA labeling beads (Amersham Biosciences) and mixed with 50 µCi [α-33P] dCTP (2500 Ci/mmol).
The mixture was incubated at 37ºC for 1-2 h. In order to eliminate non-incorporated components, Probe Quant G-50 columns (Amersham Biosciences) were used.
|
| |
|
| 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 |
After the washing step, membranes were kept humid, sealed in Saran wrap, avoiding any bubbles, and exposed to an imaging plate (BAS-MP, FujiFilm) for various times.
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 the Phosphorimager.
|
| Data processing |
Raw image quantization background subtracted
|
| |
|
| Submission date |
Dec 14, 2007 |
| Last update date |
Dec 14, 2007 |
| Contact name |
Jose E. Perez-Ortin |
| E-mail(s) |
[email protected]
|
| 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 |
GPL772 |
| Series (1) |
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