Hybridization

The two cDNA (or cDNA and gDNA) samples are purified from the labeling reaction and combined so that both are competitively hybridized to the array at the same time. Before hybridization, the cDNA/DNA samples must be denatured to single-stranded molecules with a brief incubation at 95 °C and added to the hybridization mixture (a salt and detergent solution) which provides a highly stringent environment for the binding reactions to occur in. The slide must also be prehybridized before use to prevent nonspecific binding of nucleotides. During the hybridization reaction the samples are hybridized to the array underneath a slightly raised cover slip. The entire array is then incubated within a sealed chamber, which is submersed in a water bath, for 16-20 h. The incubation temperature is set so that it is high enough to induce stringent binding conditions, the exact temperature depending on the bacterial species and the G/C content of the genome. After hybridization the microarray must be stringently washed to remove any unbound or nonspecifically bound cDNA/DNA.

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