Preparing Chromosome Spreads

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Tony Rook
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Preparing Chromosome Spreads

Please find the following protocol:

Preparing Chromosome Spreads

Procedure:

1. Expose an exponentially-growing culture of cells to Colcemid (CAUTION Biohazard!) in culture medium to arrest cells in the mitotic phase of the cell cycle. 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 μg Colcemid in 10 to 50 ml of cell culture for between 1 and 4 hours).

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 37°C.

4. Remove the KCl solution.

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

Solutions:

75 mM KCl

Fixative
3:1 Methanol:Acetic Acid

Bioreagents and Chemicals:

Potassium Chloride
Colcemid
Methanol
Acetic Acid

Reference and Links:

1. Johnston FP, Church RB, Lin CC. Chromosome rearrangement between the Indian muntjac and Chinese muntjac is accompanied by a deletion of middle repetitive DNA. Can J Biochem 1982; 60:497-506.

http://www.bio.com/protocolstools/protocol.jhtml?id=p834