|
| Status |
Public on Oct 22, 2009 |
| Title |
55 C Experiment 1 Repeat 2 Chip 13366228 Scanned at PMT Cy5 750 and Cy3 700 |
| Sample type |
other |
| |
|
| Channel 1 |
| Source name |
Experiment 1 Cy5 sample mixture
|
| Organism |
synthetic construct |
| Characteristics |
e1 cy5 sample composition:
Conc. <Set 1> <Set 2>
10-5M t1 n10 t1 n6
10-6M t2 n9 t2 n7
10-7M t3 n8 t3 n8
10-8M t4 n7 t4 n9
10-9M t5 n6 t5 n10
|
| Biomaterial provider |
Integrated DNA Technology, Inc. <http://www.idtdna.com>
|
| Extracted molecule |
other |
| Extraction protocol |
not applicable; synthesized oligonucleotide samples
|
| Label |
Cy5
|
| Label protocol |
10 ul of the 10 variably diluted oligos for Set 1 were pooled into the same tube. Similarly, 10 ul of the 10 variably diluted oligos for Set 2 were pooled into another tube. 10 ul from each tube and 1 ul of the hygromycin control at 10-7M were then taken out to be coupled with Cy5 fluorescent dye. IDT synthesized oligos included a 5' amino modifier C6 for dye incorporation. The labeled oligos were cleaned using QIAquick PCR Purification Kit (Qiagen) to discard uncoupled dyes.
|
| |
|
| Channel 2 |
| Source name |
Experiment 1 Cy3 sample mixture
|
| Organism |
synthetic construct |
| Characteristics |
e1 cy3 sample composition:
Conc. <Set 1> <Set 2>
10-5M t6 n5 t6 n1
10-6M t7 n4 t7 n2
10-7M t8 n3 t8 n3
10-8M t9 n2 t9 n4
10-9M t10 n1 t10 n5
|
| Biomaterial provider |
Integrated DNA Technology, Inc. <http://www.idtdna.com>
|
| Extracted molecule |
other |
| Extraction protocol |
not applicable; synthesized oligonucleotide samples
|
| Label |
Cy3
|
| Label protocol |
10 ul of the 10 variably diluted oligos for Set 1 were pooled into the same tube. Similarly, 10 ul of the 10 variably diluted oligos for Set 2 were pooled into another tube. 10 ul from each tube and 1 ul of the hygromycin control at 10-7M were then taken out to be coupled with Cy3 fluorescent dye. IDT synthesized oligos included a 5' amino modifier C6 for dye incorporation. The labeled oligos were cleaned using QIAquick PCR Purification Kit (Qiagen) to discard uncoupled dyes.
|
| |
|
| |
| Hybridization protocol |
The labeled oligos were pooled together and dried in a vacuum dryer at 55 C. Microarray hybridizations were conducted based on the protocol for the rice array (http://www.ricearray.org/rice_protocols.shtml). 200 ul hybridization buffer (made from 60 ul formamide, 50 ul of 20X SSC buffer, 10 ul of 2% SDS, 10 ul of 1 ug/ul hCOT I DNA, 10 ul of 1 ug/ul poly A and 1 ul of 20 ug/ul yeast tRNA filled with water to 200 ul) was used to dissolve the labeled oligos, then heated at 95 C for 30 seconds, and applied to the microarrays. Microarray slides have been pre-hybridized for 30 min with the pre-hybridization buffer (500 ul of 10 mg/ml Bovine serum albumin, 12.5 ml of 20X SSC buffer and 250 ul of 20% SDS filled with water to 50 ml). Hybridization was done using the automatic Lucidea SlidePro Hybridizer (Amersham Biosciences) and allowed to proceed for 16 hours at the hybridization temperature setting.
|
| Scan protocol |
Microarray slides were scanned by the GenePix 4100A scanner and analyzed by the GenePix Pro 6 analysis software (Molecular Devices).
|
| Description |
Refer to the series summarizing this data for more sample composition information.
|
| Data processing |
Data presented here are background subtracted intensity values for each channel from the GenePix report file supplied. Further normalization and scaling are based on multiple hygromycin controls on each chip and are explained in the corresponding publication.
|
| |
|
| Submission date |
Feb 05, 2009 |
| Last update date |
Oct 22, 2009 |
| Contact name |
Hui-Hsien Chou |
| E-mail(s) |
hhchou@iastate.edu
|
| Phone |
515-294-9242
|
| Organization name |
Iowa State University
|
| Department |
Genetics, Development and Cell Biology
|
| Lab |
Complex Computation Lab
|
| Street address |
503 Science II
|
| City |
Ames |
| State/province |
IA |
| ZIP/Postal code |
50011 |
| Country |
USA |
| |
|
| Platform ID |
GPL8161 |
| Series (1) |
| GSE14717 |
Direct calibration of PICKY-designed microarrays |
|
