Dreamtaq green pcr 1 master mix

Oxidized purines were detected using formamidopyrimidine DNA glycosylase (Fpg) (New England Biolabs, Beverly, MA, USA) digestion of total DNA [32 (link)].
The SSBs introduced by the selective cleavage by Fpg at sites of oxidized purines blocked the amplification reaction. The PCR amplification of the ND1, Ori-l, DR1, and D-loop mtDNA regions was conducted using the respective long primer sets (Table 2). The reaction mix (total volume of 20 μL) consisted of DreamTaq Green PCR 1× Master Mix (Thermo Fisher Scientific Inc, Waltham, MA, USA), 0.5 μM each forward and reverse primer, and template DNA (5 and 7.5 ng of Fpg-treated or untreated total DNA, respectively). The cycling conditions included a pre-incubation step of 10 min at 95 °C followed by 18 cycles of 15 s at 95 °C, 15 s at 60 °C, and 1 min at 72 °C. An aliquot (5 μL) of each PCR amplification was loaded on 1.3% agarose gel. Ethidium bromide-stained bands were visualized using the Gel Doc XR documentation system (BioRad Laboratories Inc., Hercules, CA, USA). Image Lab Software (BioRad Laboratories Inc., Hercules, CA, USA) was used to analyze band intensity. The value of the ratio between Fpg-treated and untreated band intensities was expressed as the percentage of the complement to 100 to improve the graphical evaluation.