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Copper Acetate Cultured to Mid-index and Transferred

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Copper Acetate Manufacturers(WSDTY) proposed Copper Acetate cultures were grown to mid-exponential phase and were transferred to an anoxic chamber. Seven milliliters of each culture was withdrawn and was added to an equal volume of RNALater (Ambion). These samples were then spun for 6 min at 5,000g; the supernatant was poured off, and the pellets were flash frozen and stored. The cultures were then shocked with 5 mM Fe(II). Samples of shocked cultures were taken at 15 min to determine the rapid transcriptional response. Many bacteria respond to stress within 15 min, and preliminary tests indicated that this held true

RNA extraction was performed with the RNeasy mini kit (Qiagen) using the digestion, lysozyme, and mechanical disruption method described in the manufacturer's protocol. Isolated RNA was treated with Turbo DNase (Ambion) by using the rigorous treatment protocol to remove contaminating DNA and was quantified by absorbance with a NanoDrop spectrophotometer.The RNA quality was confirmed on an Agilent Bioanalyzer and by checking A260/A280 and A260/A230 ratios on a NanoDrop spectrophotometer. Total RNA was amplified by using the MessageAmp II bacterial RNA kit (Ambion) according to the manufacturer's directions, with aminoallyl UTP incorporated at the final step. Cy3 dyes (Amersham/GE) were incorporated using the standard protocol. Labeled RNA was hybridized to a custom Agilent microarray, which contained 60-Per oligonucleotide probes covering all protein-coding genes and noncoding RNA features based on the R. TIE-1 genome annotation available in GenBank (accession number NC_011004). The microarray data were analyzed in R using the limma package (28). Differentially expressed genes were defined as having a P value of <0.05 after Bonferroni's correction for multiple testing.

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