Coupling Differential Centrifugation with Exonuclease Treatment and Size Exclusion Chromatography (DIFSEC) for Purification of mtDNA from Mammalian Cells

Direct analysis of mtDNA using PCR-free methods is limited by the presence of persistent, contaminating nucleic acids originating from the nuclear genome, even following stringent mitochondrial isolations. Here we describe a method developed in our laboratory that couples existing, commercially available mtDNA isolation protocols with exonuclease treatment and size exclusion chromatography (DIFSEC). This protocol produces highly enriched mtDNA extracts from small-scale cell culture, with near-undetectable nuclear DNA contamination.