Preparation ofStaphylococcus aureusGenomic DNA Using Promega Nuclei Lysis and Protein Precipitation Solutions, Followed by Additional Cleanup and Quantification Steps

Abstract

In this protocol, we describe the isolation of genomic DNA (gDNA) fromStaphylococcus aureususing the Promega Nuclei Lysis and Protein Precipitation solutions. Gram-positive bacteria such asS. aureusare harder to lyse than Gram-negative bacteria. Hence, the first step in the procedure for isolating gDNA from Gram-positive bacteria consists of a mechanical lysis step (e.g., using a bead beating grinder or homogenizer) or an enzymatic lysis step. For the method described here, the peptidoglycan layer ofS. aureusis digested with an enzyme called lysostaphin. This enzyme cleaves the pentaglycine cross-bridges within the peptidoglycan ofS. aureus.After this lysis step, the gDNA can be purified using procedures similar to those used for Gram-negative bacteria. We include additional cleanup and quantification procedures in the final steps of this protocol, in case the gDNA is subsequently used for genome-sequencing projects. By modifying the bacterial lysis step, the procedure can be easily adapted to isolate gDNA from other bacteria.