Analysis of mtDNA topology

Cells were washed once with PBS and then resuspended in lysis buffer (75 mM NaCl, 50 mM EDTA, 20 mM HEPES (pH 7.8), 0.5% (w/v) SDS and 0.2 mg/ml proteinase K). DNA was isolated by sequential phenol and chloroform extractions and resuspended in TE (pH 8). DNA (2.5 μg) was separated on 0.4% agarose gels, without ethidium bromide, at 35 V for 22 h. As controls, 1 μg of DNA from wild‐type U2OS Flp‐In cells was treated with 10 U of BamHI‐HF (NEB) or 5 U of E. coli Topoisomerase I (NEB) for 30 min at 37°C in 1 × CutSmart buffer (NEB), before being loaded on gels alongside uncut DNA as above. After electrophoresis, gels were prepared for Southern blotting as above for restricted samples.