Amersham hybond n

Freshly isolated mitochondria (800 μg) were resuspended in incubation buffer (250 mM sucrose, 100 mM KCl, 10 mM K₂HPO₄, 0.05 mM EDTA, 5 mM MgCl₂, 1 mM ADP, 10 mM glutamate, 2.5 mM malate, 10 mM Tris-HCl, pH 7.4) containing 1 mg/ml BSA, 50 μM dTTP, 50 μM dATP, 50 μM dGTP and 20 μCi [α-³³P]dCTP (3000 Ci/mmol). Samples were incubated at 37°C for 2 h on a rotating wheel. For pulse-chase experiments, mitochondria were incubated with [α-³³P]dCTP (final concentration 5 μM) for 2 h, followed by a chase (1 h) with non-radiolabeled dCTP (5 mM). Mitochondria were pelleted at 9000 g for 4 min at 4°C and washed twice with incubation buffer. Mitochondria were resuspended in 300 μL lysis buffer and DNA was isolated with the Gentra Puregene Tissue Kit (QIAGEN) according to the manufacturer’s instruction. After DNA precipitation and centrifugation, nucleic acids were dissolved in DNA hydration solution at 55°C for 1 h. DNA was analyzed by agarose gel electrophoresis. After the gel run, wet transfer was performed in 20x SSC (150 mM NaCl, 15 mM sodium citrate dihydrate) overnight onto a nylon membrane (Amersham Hybond™-N+) (Gensler et al., 2001 (link), Matic et al., 2018 (link)).