Preparing Chromosome Spreads

Monday, October 27, 2003

Description
Preparing Chromosome Spreads

Procedure
1. Expose an exponentially-growing culture of cells to Colcemid (CAUTION! see Hint #1) in culture medium to arrest cells in the mitotic phase of the cell cycle (see Hint #2).

2. Remove the Colcemid-containing Media.

3. Add 15 ml of 75 mM KCl and allow the cells to swell for 20 to 25 min at 37C.

4. Remove the KCl solution.

5. Add 15 ml of fixative and incubate the cells for 12 to 24 hr at 4C to fix.

6. Drop 2 to 3 drops of the cell/fixative solution onto a slide covered with cold ddH2O.

7. Allow the slides to air-dry and view under light microscopy or prepare for immunostaining.

Recipes
75 mM KCl

Fixative 3:1 Methanol:Acetic Acid

Supplies

Tips
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. Amount of Colcemid per given volume of cell culture and the length of incubation will depend on the cell type (a good starting point is approximately 0.1 g Colcemid in 10 to 50 ml of cell culture for between 1 and 4 hours).

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Preparing Chromosome Spreads