Expresshyb buffer

Genomic representations of all 110 Foc isolates were generated using the same complexity reduction method (PstI/HpaII), which has been developed for DArT library. Representations were precipitated with one volume of isopropanol, denatured at 95°C for 3 min, and labelled with fluorescent dye (1.5 μl of 500 μl Cy3/Cy5-dUTP, random decamers synthesized by Sigma, Australia) using the exo-Klenow fragment of E. coli DNA polymerase I (NEB, UK). For hybridization, labelled representations (also called targets) were mixed with 50 μl of DArT hybridizer (50 : 5 : 1 mixture of Express Hyb buffer (Clontech, USA), 10gL⁻¹ herring sperm DNA (Sigma-Aldrich, USA), and the 6-FAM-labeled poly-linker fragment of the plasmid that was used for library preparation, and denatured at 95°C for 3 min [14 (link)]. After denaturing labelled targets were hybridized onto the microarray surface and covered with a glass coverslip (24 × 9 × 60 mm, Menzel-Glazer, Germany). Slides were quickly placed into a 65°C water bath for overnight hybridization. After overnight hybridization at 65°C, the cover slips were removed, slides were placed into slide-racks, and washed in 4 steps: step 1:19X SSC 0.1% SDS for 5 min; step 2: in 19X SSC for 5 min; step 3: in 0.29X SSC for 2 min; and step 4: in 0.029X SSC for 30 seconds. Slides were centrifuged and dried in vacuum desiccators.