Aging Aging

114 Engel, Adibzadeh, and Pawelechand, so that the gel can be transferred rapidly into the stop bath once the reactionhas progressed far enough.5. Before cloning, 5–10 µL of the reamplified fragment should be checked in anagarose gel and the DNA content should be measured. The other 40–45 µL canthen be treated with the chloroform phenol extraction protocol.6. The enzyme content of the restriction probe should not be more than 1:10 of theentire volume, because the enzymes are diluted in a very highly concentratedglycerol solution that inhibits the enzyme activity.7. Before running an agarose gel, incubate the isolated plasmid vector with 1 U ofRNase/20 µL of probe for 10 min at 37°C to remove RNA contamination.8. Avoid too much pipetting after the ligase is added, because this enzyme is shearedvery easily and thereby loses its activity.9. A satisfactory and very uncomplicated ligation kit is provided by BoehringerMannheim. The ligation protocol can be followed as described but the incubationtime can be extended to 30–45 min instead of the 5 min given in the manufacturer’sdirections for use.10. The protocol that is described uses a laboratory equipped for radioactive methods!It would be useful to prepare the radioactively labeled probes in microfugetubes that have a cover with a screw thread to minimize risk of aerosols uponopening the tubes after heating.The polymerase should be added last, because the reaction immediately startswhen the enzyme is applied. If one dNTP is missed, every amplification will stopat the complementary nucleotide of the missing nucleotide.11. The membrane should not be washed for too extended a period, because thelabeled probes would be washed away. After that the membrane can be hybridizedagain with another probe. This process can be repeated up to 4–5 times.References1. Linskens, M. H., Feng, J., Andrews, W. H., Enlow, B. E., Saati, S. M., Tonkin, L. A.,Funk, W. D., and Villeponteau, B. (1995) Cataloging altered gene expression inyoung and senescent cells using enhanced differential display. Nucleic Acids Res.25, 3244–3251.2. Swisshelm, K., Ryan, K., Tsuchiya, K., and Sager, R. (1995) Enhanced expressionof an insulin growth factor-like binding protein (mac 25) in senescent human mammaryepithelial cells and induced expression with retinoic acid. Proc. Natl. Acad.Sci. USA 92, 4472–4476.3. Salehi, M., Hodkins, M. A., Merry, B. J., and Goyns, M. H. (1996) Age-relatedchanges in gene expression in the brain revealed by differential display. Experientia15, 888–891.4. Wu, H. C. and Lee, E. H. (1997) Identification of a rat brain gene associated withaging by PCR differential display method. J. Mol. Neurosci. 8, 13–18.5. Liang, P. and Pardee, A. B. (1992) Differential display of eucaryotic messengerRNA by means of the polymerase chain reaction. Science 257, 967–971.