Overnight growth in LB and genomic DNA isolation the following day
Extracted molecule
genomic DNA
Extraction protocol
Bacterial cultures were grown overnight from a population in 50 ml of Luria broth (LB) and genomic DNA was isolated according to standard methods (Ge & Taylor, 1992). Briefly, bacterial cells were concentrated by centrifugation, washed and suspended in isolation buffer (0.15 M Tris, 0.1 M EDTA, pH 8.0). Sodium dodecyl sulfate was added to 1% final concentration (vol/vol) and allowed to incubate for 1 hour at 55ºC or until the solution cleared. Two volumes of phenol:chloroform:isoamyl alcohol (25:24:1) were added and briefly mixed by vortexing. The resulting solution was separated by centrifugation at 12,000 x g for 15 minutes at 4ºC. The aqueous layer (top) was removed to a new tube and mixed with 2 volumes of chloroform. The mixture was separated by centrifugation at 12,000 x g for 15 minutes at 4ºC and the aqueous layer (top) was transferred to a clean tube. The aqueous layer was then extracted with at least 10 volumes of ice-cold ethanol. The precipitated DNA was spooled out of the mixture and suspended in TE (10 mM Tris/HCl, 1 mM EDTA, pH 8.0).
Label
biotin
Label protocol
Approximately 7.5 mg of purified gDNA from above was digested with the DNAfree DNase kit (ABI, P/N AM1906) using timed samples to provide a sample with a size range of between 20 and 200 bp. DNAse was inactivated with the Inactivation Reagent provided with the kit. Sample ranges were determined by gel electrophoresis on a 4-20% gradient gel (Biorad, Cat #161-1235) with Low MW Ladder (Invitrogen Cat #10821-015). Digested DNA was labeled with biotin by the addition of biotin-11-ddATP (PerkinElmer, Cat # NEL548001EA), 2 µl terminal transferase enzyme (Promega, Cat # M1875) and appropriate buffers. The digests were incubated for 2 hours at 37ºC and the labeling was quenched with 0.5M EDTA. Verification of label incorporation was accomplished by mixing the biotin-labeled DNA with Immunopure NeutrAvidin (Pierce, Cat # 31000), incubation at room temperature for 5 minutes, electrophoresis on a 4-20% gradient gel (Biorad, Cat# 161-1105EDU) and visualization. A clear shift in size distribution from unlabeled to labeled DNA was verified prior to hybridization.
Hybridization protocol
Microarrays are allowed to warm to room temperature for 30 minutes. The OligoB2 mixture (Affymetrix, P/N 900301) is heated to 65ºC for 5 minutes then mixed with the labeled and verified gDNA from above, DMSO and hybridization buffer included in the Affymetrix Hybridization, Wash and Stain kit (Affymetrix, P/N 900720). This solution is further denatured at 94ºC for 5 minutes. During this incubation the microarrays are equilibrated with prehybridization buffer and placed in the Affymetrix hybridization oven at 45ºC for 10 minutes rotating at 60 rpm. The labeled DNA is then hybridized to the microarray for 16 hours at 45ºC rotating at 60 rpm.
Scan protocol
Microarrays are washed and stained according to manufacturer’s specifications using two stain cycles and the prokaryotic washing protocol
Description
EHEC prototype strain
Data processing
Initial data analysis was performed with the Gene Chip Operating System (GCOS) suite of tools provided by Affymetrix. Additional analysis utilized the Affymetrix power tools (APT) software (http://www.affymetrix.com/partners_programs/programs/developer/tools/powertools.affx). The data analysis parameters have been established empirically using known reference genomes included on the array. Within the APT, the MAS5 algorithm (Hubbell et al., 2002; Liu et al., 2002) was utilized with the perfect match and mismatch calculations and a Tau of 0.150 to detect which probes were present or absent (command= apt-probeset-summarize -a pm-mm,mas5-detect.calls=1.pairs=1.Tau=0.150 -d FDA_ECSGa520423F.cdf -o mas5_Analysis.dir --cel-files cel_file_list.txt).
