Aging Aging

8-oxoguanine Levels in Nuclear DNA 175pacted then it is best to slowly pipet off the upper phase. The condition of theinterface varies with the type of cells used.4. Repeat step 3 with the aqueous layer.5. Transfer aqueous layer to a 15-mL centrifuge tube and measure volume. AddY mL of 6 M NaCl where Y = 0.311 × measured volume. Vortex-mix immediatelyfor 10 sec. Centrifuge for 10 min at 2000g, room temperature.6. Carefully decant supernatant into a 50-mL tube, discarding the pellet. Cool thesupernatant on ice for 15 min. Add 2 vol of ethanol (at –20°C). Invert gently to mix.Leave on ice for 10 min to aid precipitation of DNA. At this stage some of the DNAmay be clear and gelatinous, occupying a substantial volume at the bottom of thetube. The rest of the DNA may appear white and floating in the solution.7. Remove as much ethanol as possible. Wash with 20 mL of ice-cold 70% ethanol3×, removing the ethanol by aspiration. The DNA will become more compact andwhite in color after the first wash.8. Pick up the DNA pellet on a plastic pipettor tip and transfer to a microcentrifugetube. Remove as much ethanol as possible with the pipettor tip. Dry DNA undera stream of N 2 ; this will take about 5 min. If several samples are processedtogether, it is useful to have a manifold outlet for the N 2 supply.9. Add 800 µL of 40 mM Tris-HCl, pH 8.5. Pass N 2 over the sample for 1 min andthen seal the top with Nesco Film (Para Film). Slowly turn on a roller mixer at4°C overnight in the dark. Then leave for 2 h at 37°C to ensure that the DNA iscompletely dissolved. Dilute a 10 µL sample to 150 µL and read optical absorbanceat 260 nm and 280 nm in a microcuvet; the ratio of absorbance 260/280 nmis a measure of purity of DNA and should be around 1.8–1.9 (pure DNA is1.9–2.0). A more extensive purification would give even cleaner DNA but at therisk of further oxidation. Absorbance at 260 nm is used to calculate the approximateyield of DNA; 1 AU is equivalent to 50 µg/mL of double strand DNA.Generally 150–250 µg of DNA are obtained by our purification procedure.10. Store the DNA solution at –80°C under N 2 gas until required.3.2. Hydrolysis of DNAOur method is based on that of Richter et al. (3) with the exception that wefind 10× lower amount of phosphodiesterase I to be just as effective. DNA ishydrolyzed to its constituent nucleosides.1. The volume containing 75 µg of DNA is calculated for the solution prepared inSubheading 3.1., step 9 above. Allowing for enzymes and MgCl 2, calculate thevolume of buffer required for a final volume of 500 µL and place this amount in amicrocentrifuge tube on ice. Add the following:a. 3 µL of DNase (5 U/µL)b. 3 µL of alkaline phosphatase (0.25 U/µL)c. 3 µL of phosphodiesterase II (0.25 U/mL)d. 3.8 µL of phosphodiesterase I (1 U/mL)e. 10 µL of 0.5 M MgCl 2