Preparation of Staphylococcus aureus Genomic DNA Using Promega Nuclei Lysis and Protein Precipitation Solutions, Followed by Additional Cleanup and Quantification Steps
- 1Microbiology, School of Biological and Chemical Sciences, National University of Galway, Galway H91 TK33, Ireland
- 2Section of Molecular Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom
- ↵3Correspondence: a.grundling{at}imperial.ac.uk
Abstract
In this protocol, we describe the isolation of genomic DNA (gDNA) from Staphylococcus aureus using the Promega Nuclei Lysis and Protein Precipitation solutions. Gram-positive bacteria such as S. aureus are 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 of S. aureus is digested with an enzyme called lysostaphin. This enzyme cleaves the pentaglycine cross-bridges within the peptidoglycan of S. 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.
Footnotes
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From the Experiments in Bacterial Genetics collection, edited by Lionello Bossi, Andrew Camilli, and Angelika Gründling.
