This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Purification of Tetrahymena cell nuclei by unit gravity sedimentation
Materials and Instruments
nucleus (of cell) Move 1. Preparation of nuclei. For more product details, please visit Aladdin Scientific website.
PMSF
Medium Plexiglass container
2. Prepare revised media by adding a final concentration of 1 mmol/L PMSF to all revised media when mixing media vigorously under cold conditions.
3. Dilute 7.5 ml of 2% Revision Medium 1:1 with H2O.
4. Attach flexible tubing with a clamp to the center hole in the bottom of the polymethylmethacrylate Plexiglas container and the syringe with the one-pass valve, and the other side of the flexible tubing, which also has a clamp, to the three-pass valve of the syringe and the gradient generator.
5. The device is built with all tubes empty and the clamp should be clamped on one to determine the level of the gradient generator reservoir with the piston closed. 
6. fill the gradient generator reservoir with 3% and 6% revision medium, the gradient will form from 3% to 6%, so be sure to place the 3% revision medium into the mixed reservoir of the gradient generator. Do not mix the gradient yet.
7. Use the 3% Revision Medium to drive the air out of the tube between the gradient generator and the syringe, and clamp on the clamp when finished. Pour off the 3% medium in the syringe.
8. Remove air from the tube between the syringe and the plexiglass container of polymethylmethacrylate with 1% covering solution, and apply clamp when the syringe is empty.
9. Centrifuge the nuclei at 2000-3000 g for 5 minutes at 0~4℃ with a bucket-type rotary head. Resuspend in 12~20 ml of 2% Revision A medium using a Dounce homogenizer.
10. Add the homogenized nuclei into the syringe, open the clamp and let the cells and slowly flow into the container.
11. Add 2% Revision Medium to the syringe and rinse out any remaining nuclei. Clamp on the clip.
12. Inject a gradient of 3% to 6% Revision Medium into the polymethylmethacrylate plexiglass container.
13. Control the flow rate of the gradient, which should be slow enough to avoid mixing of liquids in the container.
14. When the gradient is complete, clamp on the clips.
15. Nuclei should be stabilized in the container at 0~4℃ for 15~16 hours.
16. Collect the fractions in a 50 ml conical centrifuge tube, bottom to top of the container.
17. Centrifuge the fractions at 1500 g for 10 minutes. Discard supernatant. Stir the flake precipitate and resuspend.
18. After methyl green staining, analyze the fractions by microscopy.
19. Assemble similar fractions and count nuclei for reference.