Aging Aging

Mitochondrial DNA Mutations 2717. PCR thermal cycler.8. Horizontal gel electrophoresis equipment.9. 1% Agarose gels containing ethidium bromide, 1× TAE running buffer.10. UV transilluminator11. [α- 32 P] dCTP (3000 Ci/mmol) (Amersham Life Science Products).12. Phenol (Molecular Biology grade).13. Chloroform:isoamyl alcohol (24:1 [v/v]).14. 7.5 M Ammonium acetate, sterile.15. Ethanol, 100%.16. Ethanol, 70% (v/v).17. Cerenkov counter.18. Appropriate restriction enzyme supplied with 10× reaction buffer: For the investigationof the T10010C mutation, the restriction endonuclease RsaI (BoehringerMannheim) is required.19. Heat block with variable temperature setting.20. Vertical electrophoresis system: We regularly use a 16 cm unit (SE 600) manufacturedby Hoefer (Pharmacia Biotech).21. 5% Nondenaturing polyacrylamide gel: This is made using a 30% polyacrylamidestock solution (29:1 acrylamide/bisacrylamide [w/w]) and contains 1× TBE(45 mM Tris-borate, 1 mM EDTA, pH 8.0) as the buffering component.22. 1× TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0) running buffer.23. Gel drying equipment (Bio-Rad Laboratories).24. Phosphorimage cassette and imaging system, including ImageQuant software(Molecular Dynamics).3. Methods3.1. Automated Sequencing of mtDNA1. For each PCR amplification, prepare the following reaction at room temperature:35.75 µL Sterile water5 µL 10× PCR reaction buffer5 µL 10× (2 mM) dNTPs1.5 µL 20µM forward primer1.5 µL 20µM reverse primer1 µL Template DNA (200 ng/µL stock)0.25 µL AmpliTaq Gold2. Centrifuge briefly and place in PCR thermal cycler.3. Perform 30 cycles of amplification as follows:Initial denaturation 94°C 12 min30 cycles 94°C 30 s58°C 30 s72°C 30 sFinal extension 72°C 8 min