Aging Aging

Telomeres and Replicative Senescence 6510. Isotopes: require [α- 32 P]ATP and [γ- 32 P]ATP for labeling ladder and probe,respectively. Alternatively one can use 33 P isotopes, but do not mix these differentisotopes in the same gel. 33 P is safer to handle but requires twice the exposuretime of 32 P.11. Gel apparatus: Gel cast of 15–20 cm long and thin combs (2 mm).12. Whatman 3MM paper cut out to 2.5 cm longer than gel in both length and width.3. Methods1. Isolation of genomic DNA: The isolation of the genomic DNA can be performedby any number of standard protocols in the literature. We recommend “DNAzol”DNA Isolation reagent (Molecular Research Center, Inc., 5645 MontgomeryRoad, Cincinnati, OH 45212, USA; Tel: 513-841-0900; Fax: 513-841-0080). Themain consideration in selecting a protocol should be to choose a method thatyields DNA fragments larger than 60 kb; otherwise results may be skewed (seeNote 1).2. Digestion of genomic DNA: For the digestion of 10 µg of high molecular weightDNA use the following recipe:10 µg of Genomic DNA10 µL of 10× reaction buffer2 µL of HinfI2 µL of RsaIX µL dH 2 O100 mL (final volume). Incubate at 37°C for 2–3 h.Run 2 mL of digested DNA with undigested DNA on a mini-gel to test for completionof digestions. Digestion is incomplete if there are fragments larger than 50 kb. Theamount of DNA loaded per lane must be at least 1–2 µg; a larger amount of DNAincreases the sensitivity of detection, especially for short telomeres.3. Agarose gel: Pour a 0.5% agarose/0.5× TBE gel. The gel must be at least 10 cmlong (we recommend 15–20 cm) and must be approx 3/4 cm thick. The longer gelallows good separation of large fragments of DNA and the thin combs prevent theDNA from diffusing. Run gel for a total of 750 V/h. Do not run gel faster than50 V, as this prevents good resolution of long telomere fragments. We recomend30 V, which should then be run for 25 h (for a total of 750 V/h).4. Loading DNA onto gel: Load at least 1–2 µg of DNA per lane. Labeling of 1 kband λDNA HindIII digest ladders is performed as follows in a 1.5-mL Eppendorftube. Ladders should be loaded last onto the gel to minimize exposure to radiation.0.5 µL of 1 kb ladder0.5 µLofλDNA HindIII digest ladder4 µL of 10× Klenow buffer3 µL of [α- 32 P]ATP31 µL dH 2 O (or add dH 2 O to 40 µL final volume)1 µL Klenow fragment40 µL (final volume)