Aging Aging

Telomeres and Replicative Senescence 67Incubate reaction at 37°C for 30 min, then use a quick spin column to separateunincorporated [γ- 32 P]ATP.8. Hybridization of gel: Prewarm the hybridization buffer (15 mL) to 37°C. For agel 10–20 cm in length use 15 mL (but no more than 20 mL) of hybridizationbuffer. Add 1 µL of label probe for every 1 mL of hybridization buffer. Keep at37°C while you prepare the gel.Prepare the gel for hybridization by placing the gel in a hybridization bag.Perform this by cutting open two sides of the bag, so that it opens up like a book.This method will prevent the gel from sticking to plastic that may lead to tearingof the gel. Place gel inside and seal ends of plastic bag once again with an ImpulseSealer so that there is only one opening. (There should be 1–2 cm of marginspace between the gel and the plastic bag.) Through this opening add theprewarmed hybridization buffer with the added probe. Check the seals for leaksover the Pyrex container before proceeding. Caution: The hybridization solutionwill be highly radioactive.Before sealing the open end of the bag, remove any bubbles that may be inthe bag. (Remember to do this over a Pyrex container in case of leaks.) It may behelpful in removing the final bubbles to seal the bag at the very edge, then squeezethe remaining bubbles to one edge, sealing them off with a second seal. You mustremove most of the bubbles before the second seal for this double sealing towork.9. Incubate the gel at 37°C for at least 6 h (although we recommend overnight). Forvery weak probes, 2–3 d may be necessary.10. Washing with SSC: Cut open plastic bag, remove the hybridization solution, anddispose in a proper radioactive waste receptacle. Carefully place gel in a Pyrexcontainer and add 500 mL of 0.5× SSC (prewarmed at 37°C) for 6–7 min. Repeatthe same wash two more times. Remove all SSC, then enclose the gel in SaranWrap before exposing to imaging film (see Note 5).11. Analysis: Analysis of the TRF length can be done either by densitometric scanningof the autoradiogram or by using a phosphoimager (see Note 6)4. Notes1. For the isolation of genomic DNA, we recommend using a guanidium basedmethod, such as DNAzol ® (Molecular Research Center). This method is fast(30 min) and reliable; however, we emphasize the importance of using wide-mouthtips to prevent DNA shearing during the isolation steps.2. Along with the samples, we recommend including two control DNA fragments,one from cells with long telomeres and the other from cells with short telomeres.For the long telomere source, one can use a subline of the 293 tumor, which hasstable TRF length of approx 10.5 kb. As source of short telomeres, Daudi cells,with a mean TRF length of approx 3.9 kb, can be used. Prepare a large batch ofgenomic DNA from the control cell lines, aliquot into small amounts, and usethese as standards to compare gel-to-gel variations in the sizes of TRF that mayoccur between experiments. It may be necessary to add more than 2 µg of DNA