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| Status |
Public on Sep 26, 2014 |
| Title |
BL21(DE3), adhE mutant |
| Sample type |
RNA |
| |
|
| Source name |
BL21(DE3)_adhE mutant
|
| Organism |
Escherichia coli BL21(DE3) |
| Characteristics |
strain background: B
genotype/variation: adhE mutant
|
| Treatment protocol |
The microarray experiment was performed with two serotypes of wild type E. coli and their adhE mutants: (1) K-12 strain, wild type; (2) B strain, wild type; (3) BW25113, adhE mutants; (4) BL21(DE3), adhE mutants.
|
| Growth protocol |
The E. coli strains were grown for six hours under anaerobic growth condition in glucose-containing complex medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For control and test RNAs, the synthesis of target cRNA probes and hybridization were performed using Agilent’s Low Input Quick Amp WT Labeling Kit, one color (Agilent Technology, USA) according to the manufacturer’s instructions. Briefly, each 0.2ug total RNA was mixed with WT primer mix and incubated at 65ºC for 10min. cDNA master mix (5X First strand buffer, 0.1M DTT, 10mM dNTP mix, RNase-Out, and MMLV-RT) was prepared and added to the reaction mixer. The samples were incubated at 40ºC for 2 hours and then the RT and dsDNA synthesis was terminated by incubating at 70ºC for 15min.
|
| Label |
Cy3
|
| Label protocol |
The transcription master mix was prepared as the manufacturer’s protocol (5X Transcription buffer, 0.1M DTT, NTP mix, RNase-Out, T7-RNA polymerase, and Cyanine 3-CTP). Transcription of dsDNA was performed by adding the transcription master mix to the dsDNA reaction samples and incubating at 40ºC for 2 hours. Amplified and labeled cRNA was purified on RNase mini column (Qiagen) according to the manufacturer’s protocol. Labeled cRNA target was quantified using ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE).
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| |
|
| Hybridization protocol |
After checking labeling efficiency, each 600ng of cyanine 3-labeled cRNA target were fragmented by adding 10X blocking agent and 25X fragmentation buffer and incubating at 60°C for 30min. The fragmented cRNA was resuspended with 2X hybridization buffer and directly pipetted onto assembled Agilent E.coli Oligo microarray. The arrays hybridized at 65°C for 17 h using Agilent Hybridization oven(Agilent Technology, USA). The hybridized miroarrays were washed as the manufacturer’s washing protocol (Agilent Technology, USA).
|
| Scan protocol |
The hybridization images were analyzed by Agilent DNA microarray Scanner (Agilent Technology, USA).
|
| Description |
HK110
Gene expression in B mutant type after 6hrs of growth under anaerobic condition
|
| Data processing |
Data quantification was performed using Agilent Feature Extraction software 9.3.2.1 (Agilent Technology, USA). The average fluorescence intensity for each spot was calculated and local background was subtracted. All data normalization and selection of fold-changed genes were performed using GeneSpringGX 7.3.1 (Agilent Technology, USA). Normalization for Agilent one-color method was performed, which is Data transformation. Set measurements less than 5.0 to 5.0 and Per Chip : Normalize to 50th percentage. Reliable genes were filtered by flag as following the Agilent manual.
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| |
|
| Submission date |
Apr 29, 2013 |
| Last update date |
Sep 26, 2014 |
| Contact name |
Seungwoo Hwang |
| E-mail(s) |
swhwang10@yahoo.com
|
| Phone |
82-42-879-8544
|
| Organization name |
Korea Research Institute of Bioscience and Biotechnology
|
| Department |
Korean Bioinformation Center
|
| Street address |
52 Eoeun-dong Yuseong-gu
|
| City |
Daejeon |
| ZIP/Postal code |
305-806 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL13359 |
| Series (1) |
| GSE46455 |
Comparison between wild type E. coli and adhE (alcohol dehydrogenase) mutants, grown under anaerobic condition |
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