One remarkable issue in mitochondrial DNA sequencing and variant detection is heavy nuclear DNA contamination persistent throughout the commonly used mitochondria enrichment protocols including differential and gradient centrifugations [1,2]. The chromosomes contain nuclear mitochondrial sequences , which might cause false positive or negative results in variant detection. This problem is often overcome by enrichment of the mtDNA by long-range PCR , however, such methods are prone to early-cycle PCR errors. Here, we improved existing mtDNA isolation protocols (e.g. Kennedy et al. (2013) ) and describe a detailed step-by-step guide to obtain high-quality, highly enriched mtDNA suitable for sequencing and low-frequency variant detection as well as for other sensitive applications.