taxonomy: Bacteria, Firmicutes, Bacilli, Lactobacillales, Enterococcacea lineage: ST17 year of isolation: 2005
Extracted molecule
genomic DNA
Extraction protocol
Total DNA for microarray hybridization and PCR was extracted using E.Z.N.A Bacterial DNA kit (Omega Bio-Tek Inc., Norcross, GA) with the following modifications; cell walls were digested with 1.5 mg lysozyme and 20 U mutanolysin for 20 minutes at 30°C, and DNA was eluted in ddH2O. For hybridization, DNA was broken down to 100-600 bp fragments by 1 min sonication at 2 µm amplitude.
Label
ULS-Cy5
Label protocol
Total DNA (2 µg) was fluorescent labelled using the Kreatech labeling kit with ULS-Cy5 according to the manufacturer instructions (Kreatech Biotechnologies, Amsterdam, The Netherlands), and degree of labelling (DoL) was calculated by measuring absorbance at 260 and 650 nm using a Nanodrop spectrophotometer.
Hybridization protocol
Hybridization of 4X2K CustomArrays were performed as described by the manufacturer (CustomArray Inc, Mukilteo, WA) at 52°C for 15 hours. Immediately after scanning, the arrays were stripped for 90 minutes according to the manufacturer’s protocol. Control scans were performed each time to monitor possible background of the subsequent re-hybridization. The arrays were stripped up to 6 times.
Scan protocol
Arrays were subsequently scanned using an Axon GenePix ® 4000B scanner
Data processing
Mean signal intensity was used for multiple replicate probes on array (1 to 5 probes). Arrays were normalized using quantile normalization.
