Chromosome spread

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.