|
| Status |
Public on Apr 15, 2011 |
| Title |
PET_SD_RP1 |
| Sample type |
genomic |
| |
|
| Source name |
Total sludge DNA from areas around Mathura oil refinery
|
| Organism |
soil metagenome |
| Characteristics |
isolation source: Sludge
|
| Growth protocol |
The Sphingomonas sp. strain NM-05 used in the study is a known degrader of γ-hexachlorocyclohexane (γ-HCH) and has been isolated from the vicinity of an industry India Pesticide Limited, Chinhat industrial area, Lucknow which is involved in manufacturing chlorinated pesticides for last 25 years. The strain was harvested in mineral salt medium with 0.34mM γ-HCH as sole source of carbon and energy. The strain Escherichia coli DH-5α was grown in Luria-Bertani medium to an O.D. of 0.4 at 37 degree centigrade with continuous shaking. The soil/sludge samples were collected in air tight vessels,transported on ice (4○C) to the laboratory and processed immediately for DNA isolation. At each sampling location multiple samples were collected from close (100m) to far (500m) from the industry, along the effluent channel and at the place where effluent channel from the industrial area falls into the river.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The total DNA to be used as target for microarray hybridization was isolated from soil/ sediment samples using the Mo Bio Power soil DNA kit (Mo Bio Inc., Carlsbad, CA, USA) as per manufacturer’s instructions. Before processing for DNA isolation, the soil/ sediments were mixed thoroughly using a mortar-pestle. One gm of soil/sediment was taken for DNA isolation, and during processing with the kit, lysozyme was added to facilitate the lysis of bacteria. The genomic DNA from pure bacterial cultures was isolated using the Wizard genomic DNA isolation kit (Promega corporation, Madison, USA) as per manufacturer's instructions. The concentration of DNA was analyzed by Nanodrop spectrophotometer (Nanodrop Technologies Inc, Rockland, USA), and quality was determined by analysis on DNA 12000 kit (Caliper Sciences, USA) using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
The samples for Comparative Genomic DNA Hybridization were labeled using Agilent Genomic DNA labeling Kit (Part Number: 5190-0453). DNA of each sample was digested using Alu1 and Rsa1. This restricted DNA was then labeled with Cy3 dUTP using random primer labeling method. The labeled DNA was then concentrated and quality assessed for yields
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| |
|
| Hybridization protocol |
1.25 micrograms of cy3 labeled samples were hybridized. Hybridizations were done using the aCGH Hybridization kit of Agilent (Part Number: 5190-0404). Hybridization was carried out in Agilent's Surehyb Chambers at 65º C for 24 hours
|
| Scan protocol |
The hybridized slides were washed using Agilent aCGH wash buffers (Part No: 5188-5221/22) and scanned using the Agilent Microarray Scanner G2505C
|
| Description |
Comparative genomic hybridization for profiling of biodegradation and 16S rRNA gene in petroleum contaminated site
|
| Data processing |
Images were quantified using Agilent Feature Extraction Software (version 9.5.1.1 & 10.5.1.1) and obtained background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Sep 24, 2010 |
| Last update date |
Apr 15, 2011 |
| Contact name |
Natesan Manickam |
| E-mail(s) |
nmanickam@iitr.res.in
|
| Phone |
+91-522-2620107
|
| Fax |
+91-522-2628227
|
| Organization name |
Indian Institute of Toxicology Research
|
| Department |
Department Environmental Biotechnology Division
|
| Lab |
Environmental Biotechnology Lab
|
| Street address |
Mahatma Gandhi Marg
|
| City |
Lucknow |
| State/province |
Uttar Pradesh |
| ZIP/Postal code |
226001 |
| Country |
India |
| |
|
| Platform ID |
GPL10926 |
| Series (1) |
| GSE24353 |
Development and evaluation of a 60-mer oligonucleotide microarray for profiling of biodegradation and bacterial 16S rRNA genes in diverse contaminated ecosystems |
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