
Genomic DNA isolation from Mycobacterium smegmatis has always been a challenging task due to the tough cell wall of the organism. Published methods for DNA extraction from M. smegmatis are tedious that yield only small quantities of DNA. This paper presents a simple and reproducible protocol for obtaining good quality DNA. The method is a part of a procedure used for extraction of the cell-wall-associated iron-chelating compound, mycobactin S from M. smegmatis. This method yields significant quantities of DNA as a by-product of mycobactin S extraction.