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In this protocol, we describe the isolation of genomic DNA (gDNA) from Staphylococcus aureus strains using a chloroform extraction and ethanol precipitation method. This gDNA-isolation method is well-suited for downstream whole-genome sequencing applications when working with S. aureus strains that contain plasmids, as only a small amount of plasmid DNA is isolated along with the gDNA. Similar to other gDNA isolation methods for Gram-positive bacteria, the first step in the procedure is a mechanical lysis (eg, using a bead beating grinder) or an enzymatic lysis step. In this protocol, the peptidoglycan layer of S. aureus is digested with an enzyme called lysostaphin. This enzyme cleaves pentaglycine cross-bridges within the peptidoglycan of S. aureus. After this lysis step, gDNA can be purified using similar procedures as those used for Gram-negative bacteria. We include additional cleanup and …