|
| Status |
Public on Jul 25, 2009 |
| Title |
ancestor versus CV115 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
evolved strain CV115
|
| Organism |
Escherichia coli K-12 |
| Characteristics |
parent strain: JA122
growth medium: Luria broth
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from cells grown in batch culture using a modification of methods described by Syn and Swarup [92]. Subsequent to DNA precipitation, spun pellets were re-suspended in 1XTE (10 mM Tris, 1 mM EDTA, pH 8.0) containing 50μg/mL DNAse-free RNAse A and incubated at 37°C for 30 minutes. Samples were re-extracted once with phenol:chloroform (3:1), once with phenol:chloroform (1:1) and twice with chloroform and then precipitated with EtOH using standard techniques. Following re-precipitation, the DNA was dissolved in TE.
|
| Label |
Cy5
|
| Label protocol |
CGH was performed using protocols developed at the J. Craig Venter Institute (http://pfgrc.tigr.org/protocols/protocols.shtml) with the following modifications. 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 seconds and a final concentration of 0.5 mM, and 1:1 aa-dUTP:dTTP labeling mixture was used in the Klenow reaction.
|
| |
|
| Channel 2 |
| Source name |
ancestor JA122
|
| Organism |
Escherichia coli K-12 |
| Characteristics |
growth medium: Luria broth
ancestor strain: JA122
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from cells grown in batch culture using a modification of methods described by Syn and Swarup [92]. Subsequent to DNA precipitation, spun pellets were re-suspended in 1XTE (10 mM Tris, 1 mM EDTA, pH 8.0) containing 50μg/mL DNAse-free RNAse A and incubated at 37°C for 30 minutes. Samples were re-extracted once with phenol:chloroform (3:1), once with phenol:chloroform (1:1) and twice with chloroform and then precipitated with EtOH using standard techniques. Following re-precipitation, the DNA was dissolved in TE.
|
| Label |
Cy3
|
| Label protocol |
CGH was performed using protocols developed at the J. Craig Venter Institute (http://pfgrc.tigr.org/protocols/protocols.shtml) with the following modifications. 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 seconds and a final concentration of 0.5 mM, and 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 |
evolved strains were isolated after 770 generations in a glucose limited chemostat (Davis minimal medium, 0.0125% glucose, dilution rate = 0.2/hr.)
|
| Data processing |
a-CGH images were processed using a combination of GenePix Pro 5.0, the TIGR TM4 software suite available at (www.tm4.org), and Microsoft Excel. Image analysis and spot filtering was done in GenePix and a-CGH spots were considered acceptable if they: (1) passed the default flag conditions imposed by the software during spot finding; (2) had an intensity:background ratio >1.5 and overall intensity >350 in the reference channel; and (3) had an intensity:background ratio >1.0 in the experimental channel. Ratios were normalized using total intensity normalization and replicate spots were averaged using TIGR MIDAS software.
|
| |
|
| Submission date |
Jul 23, 2009 |
| Last update date |
Jul 24, 2009 |
| 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 (2) |
| GSE17277 |
Array comparative genome hybridization of an evolved polymorphism in E. coli |
| GSE17314 |
Evolved polymorphism in E. coli |
|
