Pvuii hf

Total DNA from muscle (300 ng) as above was restricted using 10 U of BamHI‐HF or PvuII‐HF (NEB) as indicated for 1 h at 37°C. For experiments including T7 endonuclease I, 250 ng of muscle DNA was restricted using 10 U of BamHI for 30 min at 37°C, then 1 U of T7 endonuclease I (NEB) was added and the reactions were incubated for a further 30 min at 37°C. Reactions were separated on 0.6% agarose gels for 4 h at 110 V. Gels were incubated in depurination buffer (0.25 M HCl) for 20 min, followed by denaturation buffer (0.5 M NaOH, 1.5 M NaCl) for 2 × 10 min and then neutralisation buffer (0.5 M tris–HCl (pH 7.4), 1.5 M NaCl) for a further 2 × 10 min. Gels were blotted onto nylon membrane (Hybond‐N+, Amersham) overnight and then crosslinked using UV at 1200 mJ/cm². A probe (corresponding to mtDNA loci nt. 16262‐128) was synthesised by radiolabelling a PCR product using a Prime‐It II random primer labelling kit (Agilent); see Appendix Table S1 for primer sequences. Membranes were hybridised overnight at 60°C in hybridisation buffer (0.25 M phosphate buffer, 7% (w/v) SDS), then washed for 3 × 20 min with 1 × SSC containing 0.1% (w/v) SDS and imaged using a Typhoon FLA 9500 or exposed to X‐ray film.

To gauge the efficiency of the restriction enzymes (REs), 20 fmol (580 ng) of two complementary ssODNs designed to contain restriction sites for both enzymes (‘ssODN_REs_ctrl’ and ‘ssODN_REs_ctrl _rc’ in Supplementary Data 8) were annealed in NEB2 buffer (1×) in a final volume of 12 µL by denaturing at 98 °C for 5 min, then cooled at −0.1 °C/s to 20 °C (‘ssODN_REs_ctrl’ and ‘ssODN_REs_ctrl_rc’ in Supplementary Data 8). Then, 200 ng (2 µL) of annealed ssODNs was digested with either BfaI or PvuII-HF (1 U, New England Biolabs) in CutSmart buffer (1×) in a total volume of 10 µL and incubated at 37 °C for 1 h. Immediately after digestion, reactions were separated on an agarose gel (1.5%) stained with SYBR Safe (1×), imaged (UVP BioDoc-It) and analysed semi-quantitatively by gel densitometry using ImageJ (Supplementary Data 14).