Large scale DNA extraction followed by sonication at 2Am for 15min with 30sec intervals
Label
Cy3
Label protocol
Three micrograms of random primers and 10 µg of DNA were dissolved in distilled water up to the total volume of 41 µl was reached. The mixture was then centrifuged briefly, heated at 95°C for 5 min and cooled on ice for 3 min. The labeling process was performed by random-primed reaction (using hexamers) with 1.5 µl of dNTPs (5 mM dA/G/T, 2 mM dC), 1 µl of klenow fragment, exo¯ (5U/µl) and 1.5 µl (2 mM) of Cy3 dCTP (GE Healthcare, Bucks, UK) at 37°C for two hours followed by 15 min. incubation at 75°C to inactivate the enzyme. Purification of the labeling reaction was performed with Qiagen MinElute purification kit (Qiagen, Crawley, United Kingdom) according to manufacturer instructions. Purified labeled DNA (1 µl) was quantified using the NanoDrop ND1000 Spectrophotometer (MASON Technology, Dublin, Ireland) to minimize the amount of sample consumed by the measurement
Large scale DNA extraction followed by sonication at 2Am for 15min with 30sec intervals
Label
Cy5
Label protocol
Three micrograms of random primers and 10 µg of DNA were dissolved in distilled water up to the total volume of 41 µl was reached. The mixture was then centrifuged briefly, heated at 95°C for 5 min and cooled on ice for 3 min. The labeling process was performed by random-primed reaction (using hexamers) with 1.5 µl of dNTPs (5 mM dA/G/T, 2 mM dC), 1 µl of klenow fragment, exo¯ (5U/µl) and 1.5 µl (2 mM) of Cy3 dCTP (GE Healthcare, Bucks, UK) at 37°C for two hours followed by 15 min. incubation at 75°C to inactivate the enzyme. Purification of the labeling reaction was performed with Qiagen MinElute purification kit (Qiagen, Crawley, United Kingdom) according to manufacturer instructions. Purified labeled DNA (1 µl) was quantified using the NanoDrop ND1000 Spectrophotometer (MASON Technology, Dublin, Ireland) to minimize the amount of sample consumed by the measurement
Hybridization protocol
Genomic DNA hybridizations were carried out in metal chamber placed in water bath. Before hybridisation, 40 µl of purified sample was mixed with 10 µl of salmon sperm DNA (1 µg/ µl), 120 µl of pre-heated to 42°C salt-based hybridization buffer and heated for 3 min. at 95°C followed by 1 min. snap on ice. Final volume of 170 µl sample was applied on the OciChip™ array (Ocimum Biosolutions, Hyderabad, India) and hybridized for 24h at 42°C. Microarray slides were washed with pre-warmed washing buffer 1 (2x Salin-Sodium Citrate, 0.1% SDS) at 42°C on orbital shaker for 5 min, buffer 2 (1x SSC) at 30°C and buffer 3 (0.1x SSC) at 30°C. Washed slides were dried in a 50 ml conical-bottom tubes by centrifugation at room temperature for 1 min. at 500 rpm
Scan protocol
Scanned on Agilent 428 Array Scanner
Description
raw_file: replicate1_(test) = 5367_1.txt raw_file: replicate2_(test) = 5367_2.txt raw_file: replicate1_(reference) = 5367_4571_1.txt raw_file: replicate2_(reference) = 5367_4571_2.txt Four replicates of hybridization to the reference fully sequenced genome of Lb. helveticus DPC4571
Data processing
background subtracted data obtained from log2 of processed green signal/processed red signal, global normalization. Microprep and Genesis tools used
