7 protocols
normalization data transformation protocol
Microarray data was analyzed with a custom R-script in R 2.15.2. The GenePix files were imported to R, and the Limma package was used for filtering, normalization, and further analysis.
array scanning protocol
The slides were scanned with an Axon 4000B scanner using the GenePix Pro 6.0 software (Molecular Devices, CA, USA).
(Parameters: Scanning hardware = Axon- GenePix4000B, Scanning software = GenePix Pro [Axon Instruments])
nucleic acid hybridization to array protocol
Labeled DNA was mixed with hybridization solution (30 % formamide, 5×SSC, 0.1 % SDS and 0.1 mg/ml salmon sperm DNA) denatured at 95 degreesC for 2 minutes, and incubated at 42 degreesC before hybridization. Hybridization was carried out over night at 42 degreesC in a hybridization chamber (Monterey Industries, CA, USA) humidified with 5×SSC. After hybridization, the slides were washed at 42 degreesC in a buffer containing 0.5×SSC and 0.01 % SDS followed by 0.06×SSC, and finally in isopropanol before they were spun dry.
(Parameters: Chamber type = Corning Microarray Technology- CMT-Hyb chamber, Quantity of label target used = 20, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 42)
nucleic acid labeling protocol
Complementary strand DNA synthesis, labeling and purification was carried out with the FairPlay III™ microarray labeling kit (Stratagene, USA), using 500 ng random hexamers (Applied Biosystems, USA), 20 ?g of RNA, and Cy3 or Cy5 dyes (GE Healthcare Bio-Sciences AB, Sweden). After purification, the samples were concentrated with a Microcon column (Millipore, USA).
(Parameters: Amount of nucleic acid labeled = 20, Amplification = RNA polymerases, Mass unit = Micro gram)
nucleic acid extraction protocol
Bacterial cells were harvested by diluting the cultures with an equal volume of ice-cold methanol followed by centrifugation at 4,000g for 5 min at 4 degreesC. Cells were disrupted using a Precellys®24 tissue homogenizer. RNA isolation was performed using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol, including the optional on column DNAse treatment. After elution, the RNA was treated with Turbo DNAse (Applied Biosystems) according to the manufacturer’s manual followed by a second round of purification using the RNeasy Mini Kit. The concentration of RNA was determined by UV-spectrophotometry and the quality was controlled by agarose gel electrophoresis.
(Parameters: Extracted product = total_RNA, Amplification = none)
growth protocol
Single bacterial colonies of Bacilus cereus ATCC 14579 and a pBClin15 cured strain were inoculated into LB medium. (pBClin15 is a plasmid containing 28 ORFs.) The cultures were incubated at 30 C over night, diluted 1:100 in LB medium and grown to mid-log phase. These cultures were diluted to OD 0.02 in LB medium supplemnetd with norfloxacin (0.5µg /ml). The cultures were incubated for 210 min at 30 C.
(Parameters: start time = 0, stop time = 210, time unit = minutes, min temperature = 30, temperature unit = C, media = LB medium)
growth protocol
Bacteria were grown in LB medium at 30 degreesC, 220 rpm. Bacteria were inoculated from single cultures on LB plates into LB medium, incubated at 30 degreesC for 16h, diluted 1:100 in fresh LB medium and grown until mid-logarithmic phase (OD600 0.5 – 1) at 30 degreesC. These pre-cultures of logarithmically growing bacteria were used to start the experiment by diluting it to OD 0.02 in LB medium. Bacteria were incubated at 30C, 220 rpm and were harvested for RNA extraction when the cultures reached OD 0.5.
(Parameters: start time = 0, stop time = 210, time unit = minutes, min temperature = 30, temperature unit = C, media = LB medium)