|
| Status |
Public on Apr 15, 2007 |
| Title |
Drosophila_bam86_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Drosophila ovary, bam mutant
|
| Organism |
Drosophila melanogaster |
| Characteristics |
Drosophila females
Genotype: bam[delta86] mutant
Aged 3-5 days
|
| Biomaterial provider |
McKearin lab
|
| Treatment protocol |
None
|
| Growth protocol |
Standard cornmeal molasses + dry yeast
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted with TRIzol reagent
|
| Label |
Biotin
|
| Label protocol |
Standard labeling protocol
|
| |
|
| Hybridization protocol |
Full protocol at:
http://microarray.swmed.edu/AffyHybProtocol.htm
Eukaryotic Target fragmentation and Hybridization
1. Calculate the necessary amount of cRNA 20ug (cRNA concentration at least 0.625ug/ul ) Place in 0.5ml tubes. Adjust volume to 32ul with nuclease free water.
2.Add 8ul of 5x Fragmentation buffer to each sample. Vortex, spin briefly.
3. Incubate at 94?ムfor 35 minutes in the heat block.
4.While cRNA is fragmenting, take out Gene Chips and place them at room temperature.
5. . Incubate the probe array filled with 1X Hybridization Buffer at 45?ムfor 10 minutes with rotation.
Place Chips at 45?ムin Hybridization oven, rotating at 60 rpm. for 10 minutes.
6. When 35 min is up remove samples from heat block and spin down .
7.Add the following reagents
5ul of Oligo B2
15 ul of 20x Eukaryotic Controls (It is imperative that frozen stocks of 20X GeneChip Eukaryotic Hybridization Controls are heated to 65?ムfor 5 minutes to completely resuspend the cRNA before aliquotting)
3ul of herring Sperm DNA
3ul of BSA
84 ul of Nuclease free water
150 ul of 2x Hybridization Buffer
Vortex,Spin briefly..
8.. Heat the hybridization cocktail to 99?ムfor 5 minutes in a heat block.
9. Transfer the hybridization cocktail that has been heated at 99?レ to a 45?ーoven for 5 minutes.
10. Spin hybridization cocktail(s) at maximum speed in a microcentrifuge for 5 minutes to remove any insoluble material from the hybridization mixture.
11.Remove the buffer solution from the probe array cartridge and fill with appropriate volume (Table 2.2.2) of the clarified hybridization cocktail, avoiding any insoluble matter at the bottom of the tube.
12.Place probe array into the Hybridization Oven, set to 45?ヮ
Avoid stress to the motor; load probe arrays in a balanced configuration around the axis. Rotate at 60 rpm.
13.Hybridize for 16-18 hours.
|
| Scan protocol |
Standard Affymetrix scanning protocol
|
| Description |
Drosophila ovary, bam mutant
|
| Data processing |
Affymetrix GCOS
GeneSpring
|
| |
|
| Submission date |
Jan 29, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Dennis McKearin |
| Phone |
214-648-4944
|
| Organization name |
UT Southwestern Med. Ctr.
|
| Department |
Molecular Biology
|
| Street address |
6000 Harry Hines Blvd., NA5.214
|
| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390 |
| Country |
USA |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE6928 |
Stonewalling Drosophila stem cell differentiation by epigenetic controls |
|
| Relations |
| Reanalyzed by |
GSE119084 |
