|
| Status |
Public on Apr 27, 2007 |
| Title |
Isolate 7935 (7935_030) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
K10
|
| Organism |
Mycobacterium avium |
| Characteristics |
in vitro pure culture
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
QIAGEN Genomic-tip or QIAamp DNA blood kit
|
| Label |
Alexa 647
|
| Label protocol |
Purified genomic DNA was randomly sheared by nebulization on ice at 10 psi for approximately 1.5 min, resulting in an average fragment size of 800 bp. Aliquots of sheared genomic DNA (4 µg) were labeled with Invitrogen BioPrime Plus Array CGH kit.
|
| |
|
| Channel 2 |
| Source name |
7935
|
| Organism |
Mycobacterium avium |
| Characteristics |
in vitro pure culture
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
QIAGEN Genomic-tip or QIAamp DNA blood kit
|
| Label |
Alexa 555
|
| Label protocol |
Purified genomic DNA was randomly sheared by nebulization on ice at 10 psi for approximately 1.5 min, resulting in an average fragment size of 800 bp. Aliquots of sheared genomic DNA (4 µg) were labeled with Invitrogen BioPrime Plus Array CGH kit
|
| |
|
| |
| Hybridization protocol |
Labeled cDNA from experimental mycobacterial isolates was purified and mixed with alternately labeled Map K10 cDNA in a final volume of 55 µL containing 3x SSC, 0.22pct SDS, and 34 µg salmon sperm DNA (Invitrogen). This hybridization solution was incubated at 100C for 2 minutes, applied to the Map K10 microarray, and allowed to hybridize overnight at 65C. The arrays were washed sequentially for 3 minutes at room temperature in 300 mL volumes of 0.5x SSC/0.01pct SDS, 0.5x SSC, 0.1x SSC, and 0.01x SSC, then dried by centrifugation and scanned.
|
| Scan protocol |
Microarray was scanned with a ScanArray 4000 confocal laser scanner (PerkinElmer, Boston, MA). Laser and PMT power were adjusted to minimize pixel saturation and background fluorescence
|
| Description |
Comparative genomic hybridization between Map K-10 and other mycobacteria
|
| Data processing |
Raw intensity measurements for each spot on the microarray were extracted from scanned images using ScanArray Express software (PerkinElmer) and adjusted with local background subtraction and LOWESS normalization. Poorly detected spots were removed by filtering out those in which more than 50pct of the pixels from both samples were within two standard deviations of the local background. Note that for final analyses, triplicate spots were averaged and ORFs not represented by at least 2 replicate spots were discarded.
|
| |
|
| Submission date |
Feb 27, 2007 |
| Last update date |
Apr 26, 2007 |
| Contact name |
Mike Paustian |
| E-mail(s) |
mpaustia@nadc.ars.usda.gov
|
| Organization name |
National Animal Disease Center, USDA-ARS
|
| Street address |
2300 Dayton Ave
|
| City |
Ames |
| State/province |
IA |
| ZIP/Postal code |
50010 |
| Country |
USA |
| |
|
| Platform ID |
GPL3433 |
| Series (1) |
| GSE7622 |
Comparative genomic hybridizations of Mycobacterium avium isolates obtained from multiple host species |
|
