17 High Resolution FISH of Stretched Chromosome Fibers
225
Comments
1. A fixative change is necessary for preparing the lymphocyte nuclei for
the alkaline disruption. The concentration of nuclei should be at least
10
6
/ml. More (up to 10
8 ) can increase the DNA fiber recovery.
2. If only small amounts of tissue are to be digested (see comment 1) 0.20.4 ml of PK-solution are sufficient.
3. There should be no movement of the solutions on the slide. The slides
should always be in a horizontal position. Otherwise the DNA fibers are
stretched too much. The diamond circle is necessary to avoid movement of solutions on the slide.
4. The slides should always be controlled after the procedure to control
the recovery of fibers and the correct size of the fibers. If there are
enough cracked nuclei with a 'tail' of DNA fibers, finish the procedure.
5. Slides can be stored up to 1 year.
References
Arnoldus, E.P.}., Wiegant, }., Noordermeer, I.A., Wessels, }.W., Beverstock, G.C., Grosveld, G.c., van der Ploeg, M., Raap, AX (1990) Cytogent. Cell Genet. 54, 108-111
Fidlerova H., Senger, G., Kost, M., Sanseau, P., Sheer, D. (1994) Cytogenet. Cell Genet.
65,203-206
Fuchs, c., Liehr, T., Rautenstrauss, B. (1997) Trends in Genet. Online, TTO 1114
Heiskanen, M., Peltonen, 1., Palotie, A. (1996) Trends in Genet. 12, 379-382
Heng, H.H.Q., Squire, }., Tsui, L.-c. (1992) Proc. Natl. Acad. Sci. 89, 9509-9513
Liehr, T., Thoma, K., Kammler, K., Gehring, c., Ekici, A., Bathke, K.-D., Grehl, H., Rautenstrauss, B. (1995) Appl. Cytogenet. 21, 185-188
Murakami, T.) Lupski, }.R. (l996) Genomics 34, 128-133
Verma, R.S., Babu, A. (l989) Manual of Basic Techniques, pp. 5-9, Pergamon Press
225
Comments
1. A fixative change is necessary for preparing the lymphocyte nuclei for
the alkaline disruption. The concentration of nuclei should be at least
10
6
/ml. More (up to 10
8 ) can increase the DNA fiber recovery.
2. If only small amounts of tissue are to be digested (see comment 1) 0.20.4 ml of PK-solution are sufficient.
3. There should be no movement of the solutions on the slide. The slides
should always be in a horizontal position. Otherwise the DNA fibers are
stretched too much. The diamond circle is necessary to avoid movement of solutions on the slide.
4. The slides should always be controlled after the procedure to control
the recovery of fibers and the correct size of the fibers. If there are
enough cracked nuclei with a 'tail' of DNA fibers, finish the procedure.
5. Slides can be stored up to 1 year.
References
Arnoldus, E.P.}., Wiegant, }., Noordermeer, I.A., Wessels, }.W., Beverstock, G.C., Grosveld, G.c., van der Ploeg, M., Raap, AX (1990) Cytogent. Cell Genet. 54, 108-111
Fidlerova H., Senger, G., Kost, M., Sanseau, P., Sheer, D. (1994) Cytogenet. Cell Genet.
65,203-206
Fuchs, c., Liehr, T., Rautenstrauss, B. (1997) Trends in Genet. Online, TTO 1114
Heiskanen, M., Peltonen, 1., Palotie, A. (1996) Trends in Genet. 12, 379-382
Heng, H.H.Q., Squire, }., Tsui, L.-c. (1992) Proc. Natl. Acad. Sci. 89, 9509-9513
Liehr, T., Thoma, K., Kammler, K., Gehring, c., Ekici, A., Bathke, K.-D., Grehl, H., Rautenstrauss, B. (1995) Appl. Cytogenet. 21, 185-188
Murakami, T.) Lupski, }.R. (l996) Genomics 34, 128-133
Verma, R.S., Babu, A. (l989) Manual of Basic Techniques, pp. 5-9, Pergamon Press