|
| Status |
Public on May 13, 2009 |
| Title |
GCEucXylem_TimeSeries_Slide12 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
GCEucXylem_15:00
|
| Organism |
Eucalyptus camaldulensis x Eucalyptus grandis |
| Characteristics |
Light scrapings of differentiating xylem tissue. Collected from three year old Eucalyptus grandis x E. camaldulensis clone from 1-2m above ground level. Collected at 15:00.
|
| Biomaterial provider |
SAPPI Forests
|
| Treatment protocol |
Stored at -80C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
CTAB method of Zeng and Yang (2002)
|
| Label |
Cy3
|
| Label protocol |
aminoallyl labeled cDNA was prepared from 15ug total RNA using SuperScript III RT primed with random hexamers. cDNA was purified and labelled with Cy3, and excess dye removed using a Qiagen PCR Purification Kit as in TIGR protocol #M0004.
|
| |
|
| Channel 2 |
| Source name |
GCEucXylem_22:00
|
| Organism |
Eucalyptus camaldulensis x Eucalyptus grandis |
| Characteristics |
Light scrapings of differentiating xylem tissue. Collected from three year old Eucalyptus grandis x E. camaldulensis clone from 1-2m above ground level. Collected at 22:00.
|
| Biomaterial provider |
SAPPI Forests
|
| Treatment protocol |
Stored at -80C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
CTAB method of Zeng and Yang (2002)
|
| Label |
Cy5
|
| Label protocol |
aminoallyl labeled cDNA was prepared from 15ug total RNA using SuperScript III RT primed with random hexamers. cDNA was purified and labelled with Cy5, and excess dye removed using a Qiagen PCR Purification Kit as in TIGR protocol #M0004.
|
| |
|
| |
| Hybridization protocol |
Equal quantities of dye were mixed for each channel and dried in a vacuum centrifuge. Dried probes were resuspended in 60ul of hyb buffer (50%formamide, 5xSSC, 0.5%SDS, 5x Denhardt's solution, 0.5ug/ul poly d(A) and 0.5ug/ul calf thymus DNA). Denaturation for 3mins at 95C, cooled on ice and applied to pre hybridised slide and covered with cover slip. Slides were prehybridised in a (5x SSC, 1%BSA, 0.1xSDS) solution, at 42C for 45mins. Rinsed twice in water and once in isopropanol and spun dry. Scans were hybridised overnight in a water bath at 42C overnight. Post hyb washes were once in (1xSSC, 0.2%SDS at 42C) once in (0.1xSSC, 0.2%SDS at room temp) and three times in (0.1%SDS at room temp). Slides were dried and scanned within the hour.
|
| Scan protocol |
Slides were scanned on a Genepix™ 4000B scanner (Axon Instruments, Foster City, CA). Laser and photomultiplier settings were adjusted to obtain a signal ratio of 1:1 between the two dyes. Image analysis was performed with GenePix Pro 5.0 (Axon Instruments, Foster City, CA). Intensities were calculated for each spot, and spots affected by dust specks or high local background were manually flagged and removed from subsequent analysis.
|
| Description |
NA
|
| Data processing |
Raw signal intensity values (not background corrected) for each channel were transformed (log2) and the four replicate spots for each target on a single slide were averaged. The ratio of the transformed and averaged Cy3 and Cy5 values was then computed for each target.
|
| |
|
| Submission date |
Jun 10, 2008 |
| Last update date |
May 13, 2009 |
| Contact name |
Owen Luke Solomon |
| Organization name |
University of Pretoria
|
| Department |
Genetics
|
| Lab |
Forest Molecular Genetics
|
| Street address |
Lab 6-20, Agricultural Building, University of Pretoria, Lynwood Rd
|
| City |
Pretoria |
| State/province |
Gauteng |
| ZIP/Postal code |
0002 |
| Country |
South Africa |
| |
|
| Platform ID |
GPL6942 |
| Series (1) |
| GSE11731 |
Eucalyptus xylem diurnal time series |
|
