|
| Status |
Public on Dec 31, 2013 |
| Title |
BWG_butanol_3passages |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Plasmid DNA, after n-butanol selection
|
| Organism |
Escherichia coli BW25113 |
| Characteristics |
plasmid source: Post Selection Library
|
| Growth protocol |
Donor was transformed with a random genomic library made from E. coli and stored as glycerol stocks. The library was revived and transferred to four recipient strains using conjugation; the donor was miniprepped at this time, and the recipients were miniprepped after outgrowth under dual selection. Two recipient libraries in BWG and BWY were propagated under n-butanol stress for three days, and then miniprepped as well.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Library minipreps were performed with the Zyppy Miniprep kit (Zymo Technologies) and quantified using the Qubit dsDNA BR kit (Invitrogen) and the Nanodrop spectrophotometer (Thermo Scientific)
|
| Label |
Cy5
|
| Label protocol |
1 ug plasmid DNA was amplified using Exo-Klenow fragment and amino-allyl dUTP (Invitrogen) as specified by the manufacturer; the samples were then ethanol precipitated, resuspended in coupling buffer, and mixed with the appropriate CyDye (Cy3 or Cy5) according to the manufacturer's protocol. Uncoupled dye and other impurities were removed from each sample with size selective column. A NanoDrop spectrophotometer was used to assess labeling efficiency and DNA yield following this purification procedure
|
| |
|
| Channel 2 |
| Source name |
Plasmid DNA, BWG post conjugation
|
| Organism |
Escherichia coli BW25113 |
| Characteristics |
plasmid: Initial Recipient Library (BWG)
|
| Growth protocol |
Donor was transformed with a random genomic library made from E. coli and stored as glycerol stocks. The library was revived and transferred to four recipient strains using conjugation; the donor was miniprepped at this time, and the recipients were miniprepped after outgrowth under dual selection. Two recipient libraries in BWG and BWY were propagated under n-butanol stress for three days, and then miniprepped as well.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Library minipreps were performed with the Zyppy Miniprep kit (Zymo Technologies) and quantified using the Qubit dsDNA BR kit (Invitrogen) and the Nanodrop spectrophotometer (Thermo Scientific)
|
| Label |
Cy3
|
| Label protocol |
1 ug plasmid DNA was amplified using Exo-Klenow fragment and amino-allyl dUTP (Invitrogen) as specified by the manufacturer; the samples were then ethanol precipitated, resuspended in coupling buffer, and mixed with the appropriate CyDye (Cy3 or Cy5) according to the manufacturer's protocol. Uncoupled dye and other impurities were removed from each sample with size selective column. A NanoDrop spectrophotometer was used to assess labeling efficiency and DNA yield following this purification procedure
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed as specified by Agilent for array CGH 8x15k arrays; after hybridization for 24 hours, the slide was washed in aCGH buffer 1 for 5 minutes, and buffer 2 for 1 minute. The slide was immediately scanned.
|
| Scan protocol |
Scanning was performed on a GenePix 4100A scanner using the supplied software.
|
| Data processing |
Data were extracted in GenePix 4100A scanning software and normalized using the lowess method implemented in TM4 MIDAS
|
| |
|
| Submission date |
Feb 12, 2013 |
| Last update date |
Dec 31, 2013 |
| Contact name |
James Winkler |
| E-mail(s) |
jdwinkler@tamu.edu
|
| Organization name |
Texas A&M University
|
| Lab |
Kao Lab
|
| Street address |
3122 TAMU
|
| City |
College Station |
| State/province |
TX |
| ZIP/Postal code |
77843 |
| Country |
USA |
| |
|
| Platform ID |
GPL13359 |
| Series (1) |
| GSE44258 |
Construction of Reusable Plasmid Libraries for Strain Construction |
|
