Aging Aging

260 Taylor et al.2. Vortex-mix briefly, centrifuge, and aliquot 47 µL into each of eight tubeslabeled a–h.3. Add 1.5 µL of each of the appropriate forward and reverse primer.4. Centrifuge briefly and place in PCR thermal cycler5. Perform 35 cycles of amplification as follows:Initial denaturation 94°C 2 min35 cycles 94°C 30 s53°C 30 s72°C 30 sFinal extension 72°C 8 min6. Electrophorese samples (30–40 µL) through a 1.5% agarose gel for 1 h, and visualizeby UV transillumination (see Notes 17–19).4. Notes1. Ensure working solutions of oligonucleotide primers are prepared regularly, asthey are prone to degrade more rapidly at lower concentrations and after repeatedcycles of freeze–thawing.2. Identify a DNA sample that can be used as a control for each reaction; this willallow identification of nonspecific products.3. Nonspecific products can be avoided by raising annealing temperatures ordecreasing primer concentrations.4. If no product is amplified from a particular DNA sample, raise the concentrationof DNA added to the reaction up to 100 ng. If there is still no visible productcheck protein contamination and treat the sample with phenol/chloroform if necessary(this may also be required for DNA samples extracted more than a yearprior to amplification).5. If smearing occurs reduce the number of cycles or the amount of DNA added.6. The tips of the micropipets are very fragile and can be damaged during autoclaving;therefore it is important to sterilize the capillaries prior to pulling. The procedurethen needs to be performed maintaining the sterility of the micropipets.During the production of micropipets it is essential to check while measuring thediameter of the tips that a smooth edge has been produced.7. Initially using this single cell method it is recommended that a number of cellsare pooled and lysed as the template to establish the technique.8. There are several alternatives to this lysis method. Individual cells can be lysedby adding 10 µL of 200 mM KOH, 50 mM DTT, and incubated at 65°C for 1 h(21,23). Samples are neutralized by the addition of 10 µL 900 mM Tris-HCl, pH8.3, and 200 mM HCl, and the DNA phenol/chloroform extracted and precipitatedat –85°C in 2 vol of 100% ethanol, 1/10 vol of 3 M sodium acetate, pH 5.2,and 5 µL of 0.3 mg/mL glycogen (Boehringer Mannheim). The resulting pellet iscentrifuged, air-dried, and resuspended in a volume of sterile water. Alternatively,cell lysis can be performed in the PCR mix by the addition of 5 µL of 10% Triton