The widespread sampling of environmental DNA from soils has led to a fundamental shift in our understanding of Earth’s microbiome by identifying previously unknown microbes that have never been grown in the laboratory. Many of the published protocols for the extraction of environmental DNA differ based on the starting material. These differences include changes to buffer composition, detergents, and enzymatic digest. To remove this bias, we sought a standardized protocol for samples from Wind Cave National Park, which includes both above ground prairie soil and paleofill from the deepest depths of the cave system. Pre-rinsing samples with either of two solutions, 100 mM sodium phosphate pH 7.2 (Na3PO4 ) and 100mM Tris pH 8.0, 5mM EDTA, 200 mM sodium chloride (TEN), was compared and the resulting DNA was visualized using agarose gel electrophoresis. Incorporating rinses resulted in darker bands of the expected size (greater than 10 kilobases) and less DNA degradation, meaning the rinses helped increase yield while isolating more intact DNA. In the future, including a pre-lysis rinse will improve the limit of detection in cave samples that contain low microbial abundance, allowing us to better understand microbial species composition in isolated environments
