Preparation of Chromosomes

1. Autoclave heparinized tubes

2. Add 5mLs of PHA enriched RPMI-1640 Media to 15mL culture tubes and freeze.

3. Add 3-4 drops of blood to each tube (thawed) – either use heparinized tubes or drop in directly

4. Invert tubes several times

5. Incubate in CO₂ incubator (5%) at 37’C for 72hrs. keeping caps loose.

6. Every 24hrs. invert tubes

7. Add .2mL of colecimid in each tube

8. Place in incubator again for 2 hrs.

9. Centrifuge tubes at 1000rpm for 10 min.

10. Remove supernatant with vacuum pump

11. Add 5mL of hypotonic 0.075M KCl

12. Incubate tubes at 37’C, 5% CO₂ for 10 min.

13. Centrifuge at 1000rpm for 10 minutes at room temperature

14. Remove supernatant

15. Add 5mL of freshly prepared fixative

7.5mL of glacial acetic acid

22.5mL of methanol

16. Incubate tubes for 20 min. at 37’C at 4.9% CO₂

17. Remove from incubator and resuspend pellet with Pasteur pipette

18. Centrifuge at 1000rpm for 10 min.

19. Remove supernatant with vacuum pump

20. Add 5mL of fixative to pellet

21. Incubate for 20 minutes (same as 16)

22. Remove from incubator, resuspend pellet, and centrifuge

23. Remove supernatant, add fixative, and resuspend cells

Put in 4’C refrigerator