Microarray Comparative Genomic Hybridization for the Analysis of Bacterial Population Genetics and Evolution - Bacterial Pathogenesis: Methods and Protocols - page 48

BSA (400 μg/mL), either Cy5- or Cy3-labeled dUTP (2.5 nmol), and Klenow
DNA polymerase (100 U/mL) at room temperature for4htoovernight.
4. Terminate the reaction by incubation at 95 °C for 2 min. Chill on ice and add 20
m M EDTA.
5. Purify labeled genomic DNA using the Centri-spin 20 kit (Princeton Separations)
according to the manufacturer's instructions and use immediately in hybridization
experiments or store at -20 °C for later use ( see Note 5 ).
6. Determine the efficiency of labeling with an Agilent Bioanalyser or equivalent.
3.3. Slide Preparation and Pre-Hybridization
1. Add 1.8 mL of hybridization buffer (pre-warmed to 68 °C) to the slide and
incubate at 42 °C for 45 min in a hybridization chamber.
2. Wash the slide in pre-warmed (42 °C) distilled H 2 O (dH 2 O) for 30-45 s and
follow by washing in dH 2 O at room temperature.
3. Centrifuge the slide at 15 × g for 7 min. The slide is now ready for hybridization.
3.4. Hybridization and Washing
1. Add equal volumes of dH 2 O and hybridization buffer (100 μL each) to the end
reservoirs of the hybridization chamber.
2. Mix the labeled probes together with an equal amount of pre-warmed hybridization
buffer.
3. Denature the mixture at 100 °C for 5 min and add to the edge of the lifterslip.
4. Perform hybridization at 48 °C for a minimum of 2 h and as long as overnight.
5. Following hybridization, immerse the slide in 2× SSC, 0.1% SDS (pre-warmed
to 42 °C) to remove the coverslip.
6. Wash the slide twice in 0.1× SSC containing 0.1 % SDS for 10 min at room
temperature, followed by two washes in 0.1× SSC for 5 min and two washes in
0.05× SSC for 2 min.
7. Rinse the slide in dH 2 O, followed by 95% ethanol. Dry the slide by centrifugation
at 15 × g for 7 min.
3.5. Scanning of Slides and Data Analysis
1. Scan the slides using a microarray scanner such as a GenePix 4100A instrument
or equivalent with line scans performed to maximize signal and minimize
background. During scanning, normalize fluorescence intensity between channels
to a serial dilution of reference strain chromosomal DNA by adjusting laser
power and/or photomultiplier gain. Problems that have occurred throughout the
microarray experiment can become apparent upon scanning of the slides ( see
Notes 6-8 ).
2. GenePix Pro 6.0 software or similar is used to convert the pixel images into signal
intensity before exporting as a flat text file to GeneSpring (Agilent Technologies).
For each slide, adjust spot intensities for background and normalize to the median