By John R. Gosden
Transparent, step by step directions offer you the entire recommendations utilized in either classical and molecular cytogenetics. The emphasis is at the use of human fabric, however the strategies are designed to be effectively appropriate to different animal chromosomes. Chapters characteristic tools utilized in the practise of chromosomes, in reading chromosomes without delay on the light-microscope point, and in molecular genetics recommendations.
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Extra info for Chromosome Analysis Protocols
1. Transfer the cells to a lo-mL comcal bottom tube, and gently prpet the cell suspension to disperse any clumps. It is important to have a near single cell suspension (see Note 3). 2. Spin the cells at 1200 rpm for 5 min at room temperature. 3. Discard the supernatant, and tap the cells mto suspension (see Note 4). 4. Hypotomc treatment: Resuspend the cells m 7-10 mL of KCl, and leave at room temperature for 8 min. It is best to avoid any clumping at this stage also. If necessary, disperse any clumps by gently pipeting the cell suspension, Immortalized Cell Lines 55 5.
The optimal length of treatment with Triton X-100 must be found by experience. Even in the best preparations, there may be parts where the cytoplasm has not been removed sufficiently to reveal the chromosomes. However, there is no evidence that prolonged Triton treatment causes any seriousdamageto the gross morphology of the chromosomes,although it IS conceivable that it would extract chromosomal antigens. 5. It is vital that chromosome preparations are washed very thoroughly between treatments with osmium tetroxide and TCH; otherwise, the two compounds will react and form precipitates all over the specimens.
Treat with osmium tetroxide, 5 min. 4. Wash thoroughly with tap water (see Note 5). 5. Treat with thiocarbohydrazide (TCH) 5 mm. 6. Wash thoroughly with tap water (see Note 5). 7. Repeat stages 3-6 as often as needed, until the chromosomes are well blackened (up to 10 more times). (see Note 6). 8. Treat with osmmm tetroxtde, 5 mm. 9. Wash thoroughly with tap water. 10. Dehydrate through graded acetone solutions (25, 50, 75, and 100%) and critical point dry from liquid carbon dioxide. 11. Cut the slides or coverslips into small enough pieces, and attach them to the stubs with double-sided adhesive tape.
Chromosome Analysis Protocols by John R. Gosden