Nucleic acids are the basis of the biology of every organism. Numerous methodologies have been developed to extract, study and analyse DNA, as it contains information providing insights into the lifestyles and intrinsic characteristics of any organism. In the field of microbiology, DNA has been used for decades as material for studying microorganisms at the highest level of genetic resolution, i.e., at the nucleotide level. For instance, genomic DNA-DNA similarity continues to be a genotypic “gold standard” methodology for confirming whether two bacterial strains belong to the same species. Additionally, the development of next-generation sequencing technologies has enabled the rapid and cost-effective determinations of entire genome sequences of microorganisms, revolutionizing microbiological research. However, the sequencing technologies require that the DNA is extracted in a form that meets particular quantity and quality requirements.
In this protocol, we present a modified version of the Marmur procedure (Marmur, 1961), a robust and straightforward method for successful extraction, isolation and purification of total DNA from most Gram-negative and Gram-positive bacteria. The protocol provides large amounts of high-quality DNA, sufficient and suitable for most downstream applications, including DNA-DNA hybridization, DNA fragment profiling, as well as for the most widely-used next-generation sequencing technologies, i.e., Illumina (Illumina, Inc.), Ion Torrent (Thermo Fisher Scientific, Inc.), PacBio (Pacific Biosciences of California, Inc.) and MinION (Oxford Nanopore Technologies). Bacterial DNA, using this extraction protocol, has been offered as a service at the Culture Collection University of Gothenburg (CCUG) during the last 10 years.