Aging Aging

270 Taylor et al.are prefixed by 5' CAGGAAACAGCTATGACC 3' (see Tables 1 and 2). Owingto the length of these PCR primers (35–40 basepairs), we recommend that theyare hplc purified following synthesis. Stocks for PCR amplification (20 µM) arestored at –20°C (see Note 2).6. DNA thermal cycler: This is required for the initial PCR amplifications and thesubsequent cycle sequencing. To achieve a high throughput of samples, a thermalcycler capable of running 96 samples is desirable. We use the GeneAmp ® PCRSystem 9700 (Perkin–Elmer) which has a heated lid facility, thereby negating theneed for mineral oil overlay for either the PCR amplification or cycle sequencingreactions.7. Sterile water.8. 0.2 mL—MicroAmp ® Reaction tubes (Perkin–Elmer).9. Horizontal gel electrophoresis equipment for running 1% agarose gels; 1× TAE(40 mM Tris-acetate, 1 mM EDTA, pH 8.0) running buffer containing ethidiumbromide.10. UV transilluminator.11. QIAquick PCR Purification Columns (Qiagen).12. Microfuge capable of holding 1.5-mL Eppendorf tubes.13. Dye primer cycle sequencing ready reaction kits, –21 M13 and M13Rev (Perkin–Elmer).Because both of the primers used in the PCR amplification of the fragment ofinterest have M13 tails, the product can be sequenced in both (forward andreverse) directions. Store these kits at –20°C.14. Bucket microfuge capable of spinning a 96-well PCR tray.15. Ethanol, 95% (v/v).16. Glycogen, 0.3 mg/mL solution. Store at –20°C.17. Sample buffer: A 5:1 (v/v) mixture of deionized formamide and 25 mM EDTA,pH 8.0, containing blue dextran (50 mg/mL).2.2. Last Hot Cycle PCR1. Template DNA: Although initial studies are likely to be performed on total DNAextracted from a tissue homogenate, this technique is perfectly suited to the investigationof mtDNA mutations in single cells. Methods describing the isolation ofDNA from single cells are found in the preceding chapter.2. PCR amplification: The following stock solutions are required: 2 mM (10×)dNTPs (Boehringer Mannheim), 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).3. Oligonucleotide primers: These will amplify a region of the mitochondrialgenome containing the mutation of interest. In this case, a 350-basepair fragmentis amplified using L9695 (nt 9695–9717) and H10044 (nt 10044–10022). Stocksolutions (20 µM) are stored at –20°C.4. Sterile water.5. PCR tubes: 0.5-mL Thermotubes (Applied Biosystems) are recommended.6. Ice: All PCR reactions are set up on ice.