|
| Status |
Public on Mar 01, 2013 |
| Title |
Bear I versus MG1655 technical replicate 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
E. coli K12 MG1655
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: E. coli K12 MG1655
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using an SDS lysis/phenol/chloroform method described by (Syn CK, Swarup S (2000) A scalable protocol for the isolation of large-sized genomic DNA within an hour from several bacteria. Anal Biochem 278: 86-90) with slight modifications as follows: subsequent to DNA precipitation, spun pellets were treated with 50μg mL-1 DNAse-free RNAse A and incubated at 37°C for 30 min. Samples were then re-extracted once with phenol:chloroform (3:1), once with phenol:chloroform (1:1), twice with chloroform, reprecipitated and then resuspended in TE, pH 8.0.
|
| Label |
Cy5
|
| Label protocol |
Samples were labeled according to the protocol developed by the J. Craig Venter Institute (http://pfgrc.tigr.org/protocols/protocols.shtml) with minor modifications as follows. First, 5 μg of genomic DNA was sonicated to an average fragment length of 2-5 kb using a Branson Digital Sonifier at 11% amplitude for 1.1 sec. Second, a final concentration of 0.5 mM, 1:1 aa-dUTP:dTTP labeling mixture was used in the Klenow reaction.
|
| |
|
| Channel 2 |
| Source name |
Bear feces isolate B1
|
| Organism |
Escherichia coli |
| Characteristics |
strain: Bear feces isolate B1
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using an SDS lysis/phenol/chloroform method described by (Syn CK, Swarup S (2000) A scalable protocol for the isolation of large-sized genomic DNA within an hour from several bacteria. Anal Biochem 278: 86-90) with slight modifications as follows: subsequent to DNA precipitation, spun pellets were treated with 50μg mL-1 DNAse-free RNAse A and incubated at 37°C for 30 min. Samples were then re-extracted once with phenol:chloroform (3:1), once with phenol:chloroform (1:1), twice with chloroform, reprecipitated and then resuspended in TE, pH 8.0.
|
| Label |
Cy3
|
| Label protocol |
Samples were labeled according to the protocol developed by the J. Craig Venter Institute (http://pfgrc.tigr.org/protocols/protocols.shtml) with minor modifications as follows. First, 5 μg of genomic DNA was sonicated to an average fragment length of 2-5 kb using a Branson Digital Sonifier at 11% amplitude for 1.1 sec. Second, a final concentration of 0.5 mM, 1:1 aa-dUTP:dTTP labeling mixture was used in the Klenow reaction.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed using protocols developed at the J. Craig Venter Institute (http://pfgrc.tigr.org/protocols/protocols.shtml) with the following modifications: Slides were blocked (using 5X SSC, 0.1% SDS, 1% Roche Blocking Reagent) prior to hybridization as described (http://www.genomics.princeton.edu/dunham/MDhomemadeDNA.pdf) (Roche Applied Science, Mannheim, Germany).
|
| Scan protocol |
Hybridized arrays were scanned using an Axon 4000B scanner and software package GenePix 5.0 (Molecular Devices, Sunnyvale, CA).
|
| Description |
B1-2.CGH.10.31.07.DF.barbaras2co.gpr
|
| Data processing |
Images were analyzed using a combination of GenePix Pro 6.0, the freely available TIGR TM4 software suite (www.tm4.org ) and Microsoft Excel. Spots with an intensity: background ratio > 1.5 and overall intensity > 350 in the reference channel and an intensity:background ratio of > 1.0 in the experimental channel were considered acceptable for downstream processing. Local background was subtracted for each spot, the corresponding log2 ratios were normalized using total intensity normalization, and replicate spots were averaged using TIGR MIDAS. Genes that had missing data (i.e. unacceptable spots) for more than one-third of the samples were excluded from the downstream analysis leaving 3993 ORFs in the final data set. Each sample was hybridized twice and the results averaged in Microsoft Excel after processing with MIDAS. A strict cutoff of log2 ratio > 0.9 or < 0.9 was applied for the determination of gene amplifications and gene absences, respectively.
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| |
|
| Submission date |
Mar 29, 2010 |
| Last update date |
Mar 01, 2013 |
| Contact name |
Frank Rosenzweig |
| E-mail(s) |
Frank.Rosenzweig@mso.umt.edu
|
| Organization name |
University of Montana
|
| Department |
Division of Biological Sciences
|
| Street address |
32 Campus Drive, HS 104
|
| City |
Missoula |
| State/province |
MT |
| ZIP/Postal code |
59812 |
| Country |
USA |
| |
|
| Platform ID |
GPL8872 |
| Series (1) |
| GSE21115 |
Source-tracking fecal contamination by genomic analysis of Escherichia coli from human and animal hosts |
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