|
| Status |
Public on Jul 11, 2009 |
| Title |
88-4299 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Overnight bacterial culture in BHI broth.
|
| Organism |
Escherichia coli |
| Characteristics |
strain: Porcine EPEC strain O45
|
| Treatment protocol |
See the paper Variation in the Genomic islands of porcine enteropathogenic Escherichia coli strains of serogroup O45 revealed by comparative genomic hybridization and PCR by Bruant and Zhang et al. 2009
|
| Growth protocol |
See the paper Variation in the Genomic islands of porcine enteropathogenic Escherichia coli strains of serogroup O45 revealed by comparative genomic hybridization and PCR by Bruant and Zhang et al. 2009
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cultures were centrifuged at 8000 rpm for 10 minutes and the pellet was dissolved in 15 mL of 10 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 μg/mL proteinase K and 0.5% SDS. This suspension was incubated at 50oC for 2 h and DNA extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Following centrifugation for 10 min at 8000 rpm, the upper phase was removed and precipitated by adding 0.1 volume of 3 M NaOAc (pH 5.2) and 2 volumes of 99% ethanol. The DNA precipitate was then spooled out of the solution using a sterile glass rod, washed with 70% ethanol, and dissolved in 5 mL of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) buffer.
|
| Label |
Alexa Fluor 647
|
| Label protocol |
5 μg of test genomic DNA were digested with EcoRV and PstI restriction enzymes, 3 μg of which were labeled with ULYSIS Alexa Fluor 647 dye (Invitrogen, Burlington, ON). Genomic DNA form strains MG1655, Sakai and EDL933, were digested in an analogous fashion, 1 μg of each were combined and labeled with Alexa Fluor 546 dye (Invitrogen, Burlington, ON). This labeled genomic DNA mixture was then used as a reference for all hybridizations. Unincorporated dye was removed using Qiaquick PCR purification kits (Qiagen, Mississauga, ON), according to the manufacturer’s instructions, and DNA was eluted in 30 μl of 0.1x TE buffer. Labeled DNA was vacuum-dried and resuspended in 20 μl of dH2O.
|
| |
|
| Channel 2 |
| Source name |
Overnight bacterial culture in BHI broth.
|
| Organisms |
Escherichia coli O157:H7 str. EDL933; Escherichia coli O157:H7 str. Sakai; Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
referece: The three genomes contributing to the ORFs on the microarray.
|
| Treatment protocol |
See the paper Variation in the Genomic islands of porcine enteropathogenic Escherichia coli strains of serogroup O45 revealed by comparative genomic hybridization and PCR by Bruant and Zhang et al. 2009
|
| Growth protocol |
See the paper Variation in the Genomic islands of porcine enteropathogenic Escherichia coli strains of serogroup O45 revealed by comparative genomic hybridization and PCR by Bruant and Zhang et al. 2009
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cultures were centrifuged at 8000 rpm for 10 minutes and the pellet was dissolved in 15 mL of 10 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 μg/mL proteinase K and 0.5% SDS. This suspension was incubated at 50oC for 2 h and DNA extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Following centrifugation for 10 min at 8000 rpm, the upper phase was removed and precipitated by adding 0.1 volume of 3 M NaOAc (pH 5.2) and 2 volumes of 99% ethanol. The DNA precipitate was then spooled out of the solution using a sterile glass rod, washed with 70% ethanol, and dissolved in 5 mL of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) buffer.
|
| Label |
Alexa Fluor 546
|
| Label protocol |
5 μg of test genomic DNA were digested with EcoRV and PstI restriction enzymes, 3 μg of which were labeled with ULYSIS Alexa Fluor 647 dye (Invitrogen, Burlington, ON). Genomic DNA form strains MG1655, Sakai and EDL933, were digested in an analogous fashion, 1 μg of each were combined and labeled with Alexa Fluor 546 dye (Invitrogen, Burlington, ON). This labeled genomic DNA mixture was then used as a reference for all hybridizations. Unincorporated dye was removed using Qiaquick PCR purification kits (Qiagen, Mississauga, ON), according to the manufacturer’s instructions, and DNA was eluted in 30 μl of 0.1x TE buffer. Labeled DNA was vacuum-dried and resuspended in 20 μl of dH2O.
|
| |
|
| |
| Hybridization protocol |
A 70 μl hybridization solution consisting of 30% formamide, 5x SSC, 0.1% SDS, 0.1mg/ml sonicated Salmon sperm DNA, and equal amounts of test and reference labeled DNAs, each containing at least 30 pmol of incorporated dye, was denatured at 95 oC for 5 min and briefly centrifuged to collect all the contents. DNAs were then hybridized overnight (16 hours) at 42oC
|
| Scan protocol |
Arrays were then scanned with a GenePix 4000B scanner (Axon Instruments, Redwood City, CA) or ProScanArray (Perkin Elmer, Shelton, CT) and processed using GenePix Pro 5.0.
|
| Description |
88-4299
|
| Data processing |
Data obtained from E. coli O157:H7 microarrays were normalized using the Ratio-based and Lowess methods in Acuity 3.1 (Axon instruments) before analysis. The normalized data for all strains were converted into log2 (Fluor 647/Fluor 546) in Acuity 3.1 and subsequently analyzed in Microsoft Excel. Control, blank, and test spots with a mean intensity below that of the mean of all negative controls were removed from the analysis. The arithmetic mean of the remaining spots across the duplicates was taken to construct the dataset. GACK (Genomotyping Analysis by Charles Kim), was used to generate a cut off value determining the presence or absence of genes, and a dendrogram using the Euclidean distance metric with average linkage was created with tMEV v4.1.
|
| |
|
| Submission date |
Jul 10, 2009 |
| Last update date |
Jul 10, 2009 |
| Contact name |
Chad R Laing |
| E-mail(s) |
chad_r_laing@phac-aspc.gc.ca
|
| Organization name |
Public Health Agency of Canada
|
| Department |
Laboratory for Foodborne Zoonoses
|
| Street address |
Twp Rd 9-1
|
| City |
Lethbridge |
| State/province |
Alberta |
| ZIP/Postal code |
T1J3Z4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL533 |
| Series (1) |
| GSE17036 |
Variation in the Genomic islands of porcine enteropathogenic E. coli strains of serogroup O45 revealed by CGH and PCR. |
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