The isolation of nucleic acids from a biological sample is an important step for many molecular biology applications and medical diagnostic assays. This chapter describes an efficient protocol using established acidic CTAB (with a pH value of 5.0 to 6.8) based extraction method for isolation and/or purification of high molecular weight genomic DNA from a range of fresh and difficult sources from plant, animal, fungi, and soil material. This protocol is suitable for many sequencing and genotyping applications, including large-scale sample screening.