Aging Aging

112 Engel, Adibzadeh, and Pawelec5. Wet two or three pieces of Whatman 3MM paper of the same size as the gel andplace them onto the membrane. Cut paper towels to the same size and put themonto the Whatman papers to form a stack 7–8 cm high.6. Apply a glass plate onto the top and a weight of 400–500 g onto it to ensure goodcontact during the transfer.7. The RNA is transferred overnight from the gel to the nylon membrane at roomtemperature.8. After blotting, the RNA is fixed by incubating the membrane for 5 min onto aWhatman paper that is soaked with 0.05 M NaOH (RNA site up). Before storingthe filter at 4°C in cling film it should be briefly washed with 2× SSC solution.3.10.3. Probe Hybridization and LabelingHere we describe a method which uses the standard megaprime protocolfrom the Megaprime labeling kit (Amersham) (see also Notes 10 and 11).1. From 2.5 to 25 ng of the DNA are dissolved in 10 µL of ddH 2 O. Twenty-fivenanograms of the template DNA are then applied into a microfuge tube and 5 µLof the random primer are added.2. The DNA is denatured at 95–100°C for 5 min in a boiling water bath.3. The probe is collected by a short centrifugation step and then kept at roomtemperature.4. To label the DNA template the following components are added:Unlabeled dNTPs 4 µL each of dCTP, dTTP, dGTP; 2.5 mM5 µL of reaction bufferRadiolabeled dATP5 µL of [ 32 P]dATP (specific activity; 3000 Ci/mmol]2 µL of DNA polymerase I (at –20°C until needed)11 µL of ddH 2 O5. The probe is mixed gently and incubated at 37°C for 10–30 min.6. The reaction is stopped by heating the probe at 95°C for 5 min. The surplus[ 32 P]ATP can be removed on a Sephadex G50 column.7. The probe can now be stored at –20°C for 2–3 d. Further storage should beavoided because of the short half-life of 32 P.8. Prior to hybridization, the membrane is incubated for at least 15 min at 65°C orovernight in a prehybridization box with prehybridization solution.9. For the hybridization, the labeled DNA probe is denatured by heating to95–100°C for 5 min, and then directly chilled on ice. The labeled DNA probe cannow be added into the prehybridization solution at a concentration of 1 × 10 6cpm/mL and incubated at 55–65°C for at least 12 h (the higher the hybridizationtemperature chosen, the higher the stringency of the hybridization).10. After hybridization, the filters are washed by incubating for 5 min at 65°C.This procedure is repeated if necessary and checked with a hand monitor forradioactive detection (Geiger counter). The membrane is now wrapped in a nylonfilter (e.g., Saran Wrap) and the hybridization is detected by autoradiography.