sample type: Pseudomonas putida F1 treatment: 1XSSC_55 degree C
Growth protocol
Stationary phase from mineral medium with toluene
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA from pure cultures or environmental samples (10 ml pure culture or 50 ml of aquifer water) was extracted essentially as described previously (AEM vol 67. No.5 p 2354-2359). Briefly, pelleted cells were resuspended in 1 ml Tris/HCl (100 mM pH 8.0), supplemented with 100 mM EDTA, 100 mM NaCl, 1% (wt/vol) polyvinylpyrrolidone and 2% (wt/vol) sodium dodecyl sulfate and transfered to a 2 ml Lysing Matrix E tube (Qbiogene). Cells were lyzed in a Fast Prep -24 instrument (40 sec, intensity 5.5). Samples were centrifuged at 14.000 X g for 1 min at 4 C and the supernatant washed with 1 volume phenol/chloroform (1:1), centrifuged and the aqueous phase washed with 1 volume chloroform. After centrifugation, nucleic acids (aqueous phase) were precipitated with 1 volume of ice-cold isopropanol and 1:10 volume of 3M sodium acetate and purified using a Sepharose 4B spin column. Typically, two-three such column purification steps were necessary to reach a transparent solution, out of which DNA was precipitated with 3 volumes of ethanol and 1:10 volume of 3M sodium acetate. After centrifugation and washing with 80% ethanol the pellet was resuspended in 50 l of milliQ water. Quality and quantity of DNA was analyzed on 1% agarose gels and spectrophotometrically by determination of the A260/A280 ratio.
Label
Cy5
Label protocol
genomic DNA was labeled using terminal transferase (Roche) and Cy5-dUTP (GE Healthcare) following the procedure provided by the companies but incubating 4 h at 37 degree C
Hybridization protocol
For hybridization, slides (CodeLink« Activated Slides, SurModics) were inserted into the hybridization chamber (SlideBooster SB401/800, Advalytics) and covered by coverslips (ImplenGmbH). After 10 min of incubation samples were added by diffusion. Buffer AM101 (100ul per well) and AS100 (100 ul) (both from Advalytix) were used for humidity stabilizing and temperature diffusion control respectively. Hybridization was tested at temperatures between 42 and 60 degree C with increments of 2 degree C. Optimal hybridization conditions were 55 degree C for 18 h. Slides were washed subsequently in 1X SSC 0.3% SDS (5 min, 40 degree C), twice in 1X SSC (1 min each, 20 degree C), in 0.5X SSC (1min, 20 degree C), in 0.1 SSC 0.3% SDS (1 min, 40 degree C), and twice in 0.1X SSC (1min, 20 degree C).Slides were centrifuged (ArrayIt« Microarray High-Speed Centrifuge, Arrayit Corporation) and scanned using genomic DNA Microarray Scanner (Agilent Technologies) at conditions recommended by the manufacturer.
Scan protocol
Microarrays were scanned using Agilent Scanner System (G2565CA G3 upgrade, Agilent Technology) with 5 ?m resolution
Description
Water 14 genomic DNA control
Data processing
The data were analyzed with Imagene 5.0 (BioDiscovery) using default analysis settings
Reliable analysis of the catabolic gene landscape and transcriptome for pollutant degradation using a custom open source microarray with internal calibration
