Aging Aging

252 Taylor et al.13. 5% nondenaturing polyacrylamide gel.14. Whatman 3MM filter paper.15. Saran Wrap ® .16. Running buffer (1× TBE): 90 mM Tris-borate, 2 mM EDTA, pH 8.0.17. Gel dryer (Model 543, Bio-Rad Laboratories).18. PhosphorImager and ImageQuant software (Molecular Dynamics).2.4. Semiquantitative PCR of the 4977-Basepair CommonDeletion in Tissue Homogenates1. Template DNA.2. BamHI restriction endonuclease (10 U/µL) and SuRE/Cut Buffer B for restriction(10×) (Boehringer Mannheim).3. PCR amplification: The following stock solutions are required: 2 mM (10×)dNTPs (Boehringer Mannheim), 10× GeneAmp ® PCR buffer containing 100 mMTris-HCl, pH 8.3; 500 mM KCl and 15 mM MgCl 2 (Perkin–Elmer), AmpliTaq ®DNA polymerase (Perkin–Elmer). Each PCR amplification is performed in a0.5-mL thin-walled thermo-tube (Applied Biosystems).4. Oligonucleotide primers: Two pairs of primers are used to amplify a rarely deletedregion of mtDNA (wtDNA) and mtDNA containing the common deletion(mtDNA 4977 ). The primers L3108 (nt 3108–3127) and H3717 (nt 3717–3701)amplify a 610-basepair fragment corresponding to wtDNA, whereas L8282(nt 8282–8305) and H13851 (nt 13851–13832) amplify a 593-basepair fragmentcorresponding to mtDNA 4977 . Stock solutions (20 µM) are stored at –20°C.5. Thin-walled PCR tubes: 0.5-mL thermotubes (Applied Biosystems) are recommended.6. Ice: All PCR reactions are set up on ice.7. PCR thermal cycler.8. Large horizontal gel electrophoresis unit (Maxi unit, Scotlab) with well-formingcombs to produce wells of 50 µL volume.9. 1.5% Agarose gel containing ethidium bromide, 1× TAE running buffer.10. UV transilluminator.11. Digital imaging system and imaging software for PCR quantitation (AlphaImagerand AlphaEase, Flowgen).2.5. Primer-Shift PCR1. Template DNA: The isolation of total DNA from single muscle fibers and individualneurons is described in detail in Subheading 3.2.2. PCR reagents: Primer-shift PCR is performed on a Perkin–Elmer GeneAmp ®PCR System 2400 thermal cycler using AmpliTaq ® DNA polymerase (Perkin–Elmer)and the 10× reaction buffer supplied. dNTPs are made as a 2 mM (10×) stock.Thin-walled PCR tubes (0.2 mL) are also from Perkin–Elmer.3. Oligonucleotide primers: Although any combinations of mtDNA-specific PCRprimers can be used to map mtDNA deletions, we have essentially used thosedescribed by Moslemi et al. (22), in the combinations shown in Fig. 2. Thesequences of these primers are as follows: L1 (nt 1–20), L7901 (nt 7901–7920),