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===Oxidation workflow === * Thaw & mix the 1x oxidant reagent by vortexing and centrifuging at 1000 g for 5 s at room temperature. Return the thawed solution to ice. * Denature the dsDNA, add to a new 1.5 mL tube the buffer exchanged dsDNA sample, 1 M NaOH and water as detailed below: Reagent Volume per reaction 1 ug buffer exchanged dsDNA Variable Nuclease-free water up to 21.75 1 M NaOH 1.25 uL (total will be 23 uL) * Incubate the DNA denaturing solution at 37C in a shaking incubator for 30 minutes. * Snap-cool the solution by placing the tube in an ice-water bath (0C) for 5 minutes. * While the denatured DNA solution is in the ice-water bath, add 2 uL of fresh 1x oxidant reagent. Briefly vortex and centrifuge for 5s at room temperature. Replace to ice-water bath. * Incubate the oxidation reaction at 40C for 30 minutes. Briefly vortex twice, after 10 min, and after 20 min. * The solution should remain orange after the incubation. Any other color implies the oxidant reacted with contaminants in the solution. If other colors are observed, re-start the procedure from buffer exchange step. * Prepare the PreCR repair mix: Reagent Volume per reaction dNTP (10 mM) 0.4 uL 10X ThermoPol buffer 4.0 uL 100X NAD+ 0.4 uL PreCR mix 1.0 uL Water 7.95 uL * Add 13.75 uL of PreCR repair mix to each sample and incubate 37C for 30 min.
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