Hi there,
I am going to measure oxidative DNA damage marker (8-OHdG). For that, the extracted DNA needs to be digested by nuclease P1 and alkaline phosphatase. Just wondering, does anyone have detailed protocol to digest extracted DNA by nuclease P1 and alkaline phosphatase?
I would really appreciate your help and cooperation.
Regards,
Purify DNA from cell or tissue samples by a desired method or commercial DNA purification kit. Reagents needed but not supplied: Nuclease P1 3M Sodium Acetate, pH 5.2 1M Tris pH 7.5 Alkaline Phosphatase Zinc Chloride For digestion, 15 µg of DNA in 100µL DNA hydration buffer or DI water is required. Protocol volumes can be scaled. 1. Prepare working solution of Nuclease P1 at 5U/mL in 40mM sodium acetate. Keep on ice. Remove aliquot of alkaline phosphatase, 10U/mL, from -20˚C. Keep on ice. Thaw normalized DNA samples (15 µg/100 µL). 2. Denature the DNA at 95-100˚C for 10 min. Cool completely on ice 5 min. Centrifuge for 5 sec or tap any condensate down into tube. Add 50 µL 40 mM sodium acetate pH 5.0-5.4, 0.4 mM ZnCl2 . 3. Add 50 µL of 5U/mL Nuclease P1. Invert tube to mix. Centrifuge 5 seconds or tap any condensate down into tube. Incubate at 37˚C for 30 min. 4. Adjust pH to 7.5-8.0 by adding 20 µL 1M Tris pH 7.5 to tube. Add 15 µL of 10U/mL alkaline phosphatase. Invert to mix. Centrifuge 5 sec or tap any condensate down into tube. 5. Incubate at 37˚C for 30 min. Boil samples for 10 min at 95˚C to inactivate alkaline phosphatase. Place samples on ice. Aliquot samples, 2 µg/tube and store at ≤ -20˚C until assaying. Samples should be diluted ≥ 1:4 with the diluted Assay Buffer prior to running in the assay.
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