Virtualab Protocols (Chang Bioscience)
1. Add colcemid or colchicine to 0.5 mM
to rapidly growing cells.
2. Cells accumulate in metaphase after 2-8 hours.
3. Harvest and transfer cells to a 15 ml tube.
4. Pellet cells.
5. Resuspend cells in 10 ml of hypotonic saline (e.g., growth medium
diluted 1:4 with ddH2O, or 1% sodium citrate,
or 75 mM KCl).
6. Allow cells to swell for 15-20 minutes at 37 °C.
7. Pellet cells (500 g) and then resuspend cells in 5 ml fixative solution
(e.g., cold (-20 °C) methanol:acetic acid 3:1).
8. Fix for 5 minutes.
9. Prewet the slide.
10. Place a slide at a 45° angle, and drop 1-3 drops of cell suspension onto the slide
from a height of 10 cm or more.
11. Air dry.
12. For viewing under microscopy, allow the slide to air-dry thoroughly.
Mount and cover with a coverslip, then examine under the 100X oil immersion
objective.
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