Eco105I (SnaBI) (10 U/µL)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For the ND1+ND4− PCR product, the template DNA was restricted with the Eco105I* enzyme (Thermo) to prevent WT mtDNA amplification.	All products were resolved on 0.5% 1X TBE agarose, gel purified (Qiagen, Germantown, MD), and quantitated by OD260 (Nanodrop 1000, Thermo).

Protocol tips
For the ND1+ND4− PCR product, the template DNA was restricted with the Eco105I* enzyme (Thermo) to prevent WT mtDNA amplification.
Downstream tips
All products were resolved on 0.5% 1X TBE agarose, gel purified (Qiagen, Germantown, MD), and quantitated by OD260 (Nanodrop 1000, Thermo).

Publication protocol

To generate reference PCR products, LongAmp 2X Mastermix (NEB, Ipswich, MA) was used in a 25 μL final reaction volume and PCR amplification was performed in a ProFlex thermal cycler (Applied Biosystems, Waltham, MA) with the following PCR amplification profile: 94 °C for 30 sec; 30 cycles of 94 °C for 20 sec, 55 °C for 20 sec and 65 °C for 8 min; and 65 °C for 10 min. Template concentration was 1 ng/reaction while primer concentration was at 4 μM. The double-stranded linear PCR product containing the ND1 and ND4 targets used in Fig. 3 was generated from total HeLa DNA using the hND1f and hND4r primers. The ND1+ND4+ and ND1+ND4− PCR products used in Fig. 4 were generated by PCR using total DNA isolated from HeLa (ND1+ND4−) and KSS patient (carrying the common deletion or ND1+ND4− sequence) cells and hND1f and hND6r primers. For the ND1+ND4− PCR product, the template DNA was restricted with the Eco105I* enzyme (Thermo) to prevent WT mtDNA amplification. All products were resolved on 0.5% 1X TBE agarose, gel purified (Qiagen, Germantown, MD) and quantitated by OD260 (Nanodrop 1000, Thermo).

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Manufacturer protocol

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