|
| Status |
Public on Sep 02, 2010 |
| Title |
CO92_Cy5_PestoidesF_Cy3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
PestoidesF
|
| Organism |
Yersinia pestis |
| Characteristics |
strain: PestoidesF
|
| Biomaterial provider |
Mikeljon Nikolich, Walter Reed Army Research Institute (WRAIR)
|
| Treatment protocol |
None
|
| Growth protocol |
Cells grown in Lysogeny broth (LB) at 37 degrees Celcius overnight.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated by lysozyme and proteinase K treatment.
|
| Label |
CY3
|
| Label protocol |
4ug of genomic DNA was labeled at 37 degree Celcius overnight using DNA polymerase I Klenow fragment (New England Biolabs), a 25X 2:1 amino allyl dUTP (Ambion) dNTP mix, and random hexamers (Invitrogen). Labeled DNA was purified with Qiaquick PCR purification kit (QIAGEN).The purified labelled DNA was then dye coupled with the appropriate cye dye (CY3/CY5).
|
| |
|
| Channel 2 |
| Source name |
CO92
|
| Organism |
Yersinia pestis |
| Characteristics |
strain: CO92
|
| Biomaterial provider |
Mikeljon Nikolich, Walter Reed Army Research Institute (WRAIR)
|
| Treatment protocol |
None
|
| Growth protocol |
Cells grown in Lysogeny broth (LB) at 37 degrees Celcius overnight.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated by lysozyme and proteinase K treatment.
|
| Label |
CY5
|
| Label protocol |
4ug of genomic DNA was labeled at 37 degree Celcius overnight using DNA polymerase I Klenow fragment (New England Biolabs), a 25X 2:1 amino allyl dUTP (Ambion) dNTP mix, and random hexamers (Invitrogen). Labeled DNA was purified with Qiaquick PCR purification kit (QIAGEN).The purified labelled DNA was then dye coupled with the appropriate cye dye (CY3/CY5).
|
| |
|
| |
| Hybridization protocol |
Equal concentrations of DNA probe from the appropriate Cy3 and Cy5 labeled probes were combined, dried and then resuspended in hybridization buffer as defined by the Agilent Oligonucleotide Array-Based CGH protocol. Resuspended probes were denatured at 95 degree Celcius and held at 37 degree Celcius prior to hybridization. The probe mixture then was added to the Agilent microarray slide and allowed to hybridize for 24 hours rotating at 20 rpm in a hybridization oven at 65 degree Celcius. Hybridized slides were washed sequentially as defined by the Agilent Oligonucleotide Array-Based CGH protocol.
|
| Scan protocol |
Scanned on Axon GenePix 4000 scanner. PMT values were optimized during scanning to balance channel intensities.
|
| Data processing |
log2 ratio of QUERY_MEDIAN_INTENSITY/REF_MEDIAN_INTENSITY from from default normalized Agilent Feature Extraction settings for 2 channel arrays
|
| |
|
| Submission date |
Aug 20, 2010 |
| Last update date |
Sep 01, 2010 |
| Contact name |
John Braisted |
| E-mail(s) |
jbraisted@jcvi.org
|
| Organization name |
J Craig Venter Institute
|
| Department |
PFGRC
|
| Lab |
PFGRC_EXTSW
|
| Street address |
9704 Medical Center Dr
|
| City |
Rockville |
| State/province |
MD |
| ZIP/Postal code |
20850 |
| Country |
USA |
| |
|
| Platform ID |
GPL10805 |
| Series (1) |
| GSE23797 |
Identification of new genomic areas within the genomes of Yersinia pestis and Yersinia pseudotuberculosis strains through targeted genome sequencing |
|
