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Isolation of Mitotic Chromosomes from Hela S3 Cells

Adopted from Renzi et al, 1997

 

SOLUTIONS

Swelling Buffer- 10 mM HEPES, 40 mM KCl, 5 mM EGTA, 4 mM MgSO4, pH 7.4, + Protease Inhibitors

 

Extraction/ Lysis Buffer- 60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 4 mM MgSO4, pH6.9, +1mM DTT + 1% CHAPS + Protease Inhibitors

 

1. Grow Hela S3 cells in suspension (preferably in a spinner flask) to a density of 5 x 105 cells/ ml

2. Add colcemid to a final concentration of 0.15 ug/ml for 16-18 hour

3. Spin down cells at 300g for 5 minutes (1200rpm in CR4.12 rotor)

4. Rinse pellet 2 times in swelling buffer

5. Pipette pellet up and down rigorously in 10 ml of lysis buffer

6. Spin at 64g for 5 minutes. Keep supernatant containing chromosomes (560 rpm in 15 ml conical, CR4.12 rotor)

7. Repeat steps 5 and 6 4 times

8. Spin the collected supernatants at 200g for 7 minutes to pellet interphase nuclei and cellular debris (990 rpm)

9. Spin the supernatant at 1600g for 10 minutes to pellet mitotic chromosomes (2800 rpm)

10. Wash pellet 3x in extraction/lysis buffer

11. Re-suspend in a couple ml's of same buffer

12. Layer onto a 40/80% glycerol step gradient cushion and spin at 2700g (Use HL-4 swinging bucket rotor)

13. Collect chromosomes from the interphase of the step gradient

14. Wash two times in buffer and flash freeze

 

 

This page last modified 08/11/2011

The Stukenberg Lab and the Burke Lab are in the Department of Biochemistry and Molecular Genetics at the University of Virginia