Microarray Comparative Genomic Hybridization for the Analysis of Bacterial Population Genetics and Evolution - Bacterial Pathogenesis: Methods and Protocols - page 46

2.3. Slide Preparation and Pre-Hybridization
1. Microarray hybridization chamber (Telechem, Sunnyvale, CA).
2. Hybridization buffer, ULTRAhyb (Ambion, Austin, TX), pre-heated to 68 °C.
2.4. Hybridization
1. 20× sodium chloride sodium citrate (SSC): 3.0 M NaCl, 0.3 M Na-citrate, pH 7.0.
2. 2× SSC, 0.1× SSC, and 0.05× SSC in nuclease-free water. Store at room temper-
ature.
3. 0.1% (w/v) SDS. Store at room temperature.
4. 95% ethanol, molecular grade.
5. Lifterslip (Fischer Biosciences, Rockville, MD).
2.5. Scanning of Slides and Data Analysis
1. GenePix 4100A microarray scanner or equivalent and GenePix Pro 6.0 software
(Axon Instruments, Foster city, CA) or similar.
2. GeneSpring software (Agilent Technologies).
3. Microarray validation system ( see Note 3 ).
2.6. Phylogenetic Analysis
1. Cluster and TreeView programs (11) .
3. Methods
The major steps in the CGH protocol include genomic DNA isolation and
labeling, microarray hybridization, scanning of slides and calculation of fluores-
cence intensity ratios, and data analysis by use of appropriate statistical methods
( see Fig. 1 ).
3.1. Genomic DNA Isolation and Digestion
1. Perform genomic DNA isolation from S. aureus test and reference strains using
a modification of the Edge Biosystems genomic DNA isolation kit. The modifi-
cation consists of addition of lysostaphin (100 μg/mL final concentration) to
bacterial cells and incubation at 37 °C for 10 min prior to DNA extraction.
2. Determine the quality of DNA on a Bioanalyser or equivalent; the A 260/280 nm
absorbance ratio should be >1.8. The procedure outlined is optimal for 400 ng of
staphylococcal genomic DNA.
3. Digest genomic DNA with the restriction enzyme Hin P1I at 37 °C overnight and
purify before using as a template for labeling.