BclI (10 U/µL)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	A total of 5 μg of mitochondrial nucleic acid was digested according to the manufacturer’s recommendation with BclI or HincII (both from Thermo Scientific) and run on a 0.4% agarose gel in 1× TBE until the fragments of interest had migrated 10 cm into the gel. The gel slab was rotated 90°, and a 0.95% agarose gel was cast around it.

Protocol tips
A total of 5 μg of mitochondrial nucleic acid was digested according to the manufacturer’s recommendation with BclI or HincII (both from Thermo Scientific) and run on a 0.4% agarose gel in 1× TBE until the fragments of interest had migrated 10 cm into the gel. The gel slab was rotated 90°, and a 0.95% agarose gel was cast around it.

Publication protocol

A total of 5 μg of mitochondrial nucleic acid was digested according to the manufacturer’s recommendation with BclI or HincII (both from Thermo Scientific) and run on a 0.4% agarose gel in 1× TBE until the fragments of interest had migrated 10 cm into the gel. The gel slab was rotated 90°, and a 0.95% agarose gel was cast around it. The second dimension was run until the fragment of interest had migrated ∼10 cm. For long-range 2D AGEs, 5 μg of mitochondrial nucleic acids was digested with FastDigest BamHI or PvuII (Thermo Scientific), which cut mtDNA only once, and run on a 0.28% agarose gel in 1× TBE until the linearized mtDNA fragment had migrated 10 cm into the gel. The gel slab was rotated 90°, and a 0.58% agarose gel was cast around it. The second dimension was run at 2.6 V/cm until the fragment of interest had migrated ∼10 cm from the top. Southern blotting was performed by using the standard procedure, and the nucleic acid hybridization was performed by using a PCR probe spanning nucleotides 14,783–15,333 (Cytb) of the mouse mtDNA and 4,470–5,511 (ND2) or 35–611 (NCR) of the human mtDNA. Probes were labeled by using a Rediprime II random prime labeling kit (GE Healthcare) and [α-32P]dCTP (3,000 Ci/mmol; PerkinElmer). The autoradiographs were exposed on a Kodak MS film (Sigma-Aldrich) or were captured on a Kodak storage phosphor screen SO230 and detected by using Molecular Imager FX (BioRad).

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