genotype: Wild Type phenotype: normal accession: U00006 hybridization buffer (formamide content): 10% formamide hybridization buffer (total rna): 50 ng
Growth protocol
E. coli was grown to mid-log phase (O.D.600nm 0.3-0.4) in Lennox LB broth (Becton Dickinson, Franklin Lakes, NJ) at 37°C. R. sphaeroides was phototrophically grown to mid-log phase (100-150 kletts) in Sistrom's minimal medium (Sistrom, W. 1960. Journal of General Microbiology. 22:778-785) amended with 33.9 mM succinate .
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted using Ultra Clean Soil DNA (MoBio Laboratories, Solana Beach, CA).
Label
Cy3
Label protocol
Cloning protocol: For each organism, 16S rRNA gene was amplified with 27f and 1492r primers and cloned using TOPO10 cloning kit (Invitrogen, Carlsbad, CA). The clones were sequenced to find operons that matched the GenBank sequences in accession codes used during probe design. One matching clone for each organism was subsequently re-amplified with 27f and 1492r primers and purified using a QIAquick spin column (Qiagen, City, State) for the next step. Cloned and purified 16S rRNA genes were Cy3-labeled using random prime amplification with Klenow fragment and a decalabel DNA labeling kit (Fermentas, St Leon-Rot, Germany). A previously published protocol was followed (Loy, A. et al. 2011. PCR mutation detection protocols. Methods in Molecular Biolology. Vol. 688, pp. 187-206).
Hybridization protocol
5 ng or 50ng of labeled target was hybridized with the microarray in 10 microliters of custom hybridization buffer (1M Na+, 20 mM Tris [pH=7.2], 0.02% SDS, and variable amounts of formamide) using a Nimblegen mixer matching the arrays used (see www.nimblegen.com).
Scan protocol
Scanning was performed with an Axon 4000B laser scanner and GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA). The wavelength and PMT gain were set at 532 nm and 430, respectively. Two lines were averaged. Fluorescence data was saved in TIFF files and processed using Nimblescan software (see www.nimblegen.com).
Description
Hybridization of labeled and fragmented E. coli 16S rRNA gene with 10% formamide. Total RNA in hybridization buffer is 50 ng.
Data processing
Raw data (.pair files) was analyzed using Matlab (The MathWorks, Natick, MA). For each probe, the average intensity of three replicates were calculated after the elimination of outliers based on a standard deviation test. The average of control (Nonsense) probes was subtracted from all averages to obtain background-corrected results.
