Aging Aging

250 Taylor et al.drial genome using a forward primer L3200 (nt 3200–3219) and a reverse primerH16215 (nt 16215–16196), numbered according to the Cambridge sequence (3). Forwhole genome amplification, a number of articles have been published with primersequences; we have successfully used those described by Kovalenko and colleagues(26). Stock solutions (10 µM) of primers are stored at –20°C (see Note 1).5. DNA template: Only 10–50 ng of total DNA is required for amplification of thegenome in part or whole. The quality of the DNA template is crucial for successfulamplification, and protein contamination (determined by measuring the A 260 /A 280 ratio should be no less than 1.8. We recommend that solutions of DNA(10–50 ng/µL) be made fresh from concentrated DNA stocks, although these maybe stored for 3–4 d at 4°C and for 1–2 wk at –20°C (see Note 2).6. Sterile water.7. Ice: The reaction should always be set up on ice to avoid hot start.8. Mineral oil if required to overlay the reaction.9. Thin-walled PCR tubes: These permit a more efficient transfer of heat, and assuch are crucial for amplifying long templates. Use 0.2- or 0.5-mL thermotubes(Applied Biosystems) depending upon thermal cycler used.10. Thermal cycler: Both the Hybaid Omnigene and Perkin–Elmer GeneAmp ® PCRSystem 2400 thermal cyclers give good, reproducible results.11. Horizontal gel electrophoresis equipment.12. Agarose gel containing ethidium bromide; 1× TAE (40 mM Tris-acetate, 1 mMEDTA, pH 8.0) running buffer.13. UV transilluminator.2.2. Single-Cell PCR1. Tissue sections: Fresh muscle and neuronal tissue are frozen in liquid nitrogencooled isopentane and stored at –85°C. Fresh frozen sections (30 µm) are cutusing a Brights OTF cryostat and air-dried at room temperature for 30 min. Thesesections can be used immediately or stored at –80°C in air-tight slide containers.2. Histochemical staining: The assay of COX activity requires stock solutionsof 5 mM 3,3'-diaminobenzidine tetrahydrochloride (light sensitive) (DAKO) in0.1 M phosphate, pH 7.0, and 500 µM cytochrome c (light sensitive) (Sigma) in0.1 M phosphate, pH 7.0. The assay of succinate dehydrogenase (SDH) activityrequires stock solutions of 1.875 mM nitroblue tetrazolium (Sigma), 1.30 Msodium succinate (Sigma), 2 mM phenazine methosulfate (light sensitive)(Sigma), and 100 mM sodium azide (BDH) all in 0.1 M phosphate, pH 7.0.Toluidine blue staining is performed with a 1% solution of toluidine blue (lightsensitive) (Sigma) in 1% sodium borate (Sigma).3. Capillaries: Standard wall borosilicate glass capillaries without filament, 1.0 mmouter diameter × 0.58 mm inner diameter (GC100–15 Clark ElectromedicalInstruments) are used for muscle dissection. Standard wall borosilicate glass capillarieswithout filament, 1.5 mm outer diameter × 0.86 mm inner diameter(GC150–10 Clark Electromedical Instruments) are used for neuronal dissection.Micropipets are produced using a Narishige PC-10 micropipet puller.