DNA extraction was performed according to the method described by Dirk Gevers, Geert Huys, Jean Swings (2001) Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiology Letters 205 (1), 31-36.
Label
Cy3
Label protocol
DNA samples were labeled using an adapted protocol based on the BioPrimeTM DNA Labeling System (Invitrogen). 20 µL of the 2.5x mix of random primer and reaction buffer was added to 2 µg DNA in a volume of 21 µL. The mixture was incubated at 90C for 5 min and immediately put on ice. 5 µL of a dNTP mix (1.2mM dATP, 1.2 mM dGTP, 1.2 mM dTTP, and 0.6 mM dCTP), 3µL 1mM Cy3TM dCTP (GE Healthcare), and 1 µL of Klenow fragments were added. The mixture was incubated at 37C for 16 h in a thermal cycler. The reaction was stopped by adding 5 µL of Stop Buffer. The amplification product was purified using the MinElute PCR Purification Kit (Qiagen) and the concentration was measured using NanoDrop.
Hybridization protocol
Hybridization mixtures were prepared using 40 pmol labeled DNA in 210 µL Hybridization Buffer (GE Healthcare) containing 50% formamide (Sigma-Aldrich). The hybridization mixtures were denaturized at 96C for 3 min, put on ice for at least 5 min, and were hold at 32C for 5 min and subsequently centrifuged at 12,000 rpm for 5 min. The samples were hybridized with an HS 4800 Pro Hybridization Station (Tecan Systems Inc, San Jose, USA) at 32C for 16 h. Post-hybridization washing (automated) was performed in 1x SSC / 0.2% SDS at 32C, 27C and 23C for 20sec, 20sec and 30sec, respectively. This was followed by 0.1x SSC / 0.2% SDS for 1 min at 23C and in 0.1x SSC at 23C for 30 sec. Slides were dried for 2 min at 30C using nitrogen gas.
Scan protocol
Slides were scanned using the Agilent scanner (Agilent) at 10 micron and images were analysed using ArrayVision v7 (GE Healthcare).
Description
Series_summary
Data processing
We apply a background correction, i.e. we subtract the local background for the local foreground intensities. Data are base 2 log transformed and for each clone averaged over the 4 replicates on the array.
We apply a background correction, i.e. we subtract the local background for the local foreground intensities. Data are base 2 log transformed and for each clone averaged over the 4 replicates on the array.
PRESENT.CALL
the number of times a gene was called present, i.e. the foreground intensity is above the background intensity plus three times the average of the local standard deviation of the foreground and background pixels. Each clone has four repeats, hence this number varies between 0 and 4.
