Bitan:Electron microscopy

From OpenWetWare

Jump to: navigation, search

Electron Microscopy

Glow-discharging grids

1. Clean the jar.
2. Adjust the height of the sample holder.
3. Assemble the circular electrode in the chamber over the holder.
4. Place grids in the center of the holder, close the chamber.
5. Switch the button to HTS.
6. Turn off (tight) the gas release.
7. Turn on the pump unit.
8. Switch the button to glow.
9. The glow time is 60s by default.
10. Click "Main" button.
11. Release the gas carefully to keep the pressure at 60.
12. Press "START"
13. Turn the charge switch to 4-5.
14. Sixty sec later turn the charge back to 0.
15. Release the gas.
16. Turn off the Main button.
17. After the gas is balanced, release the jar and take out the grids.
18. Put the jar back.
19. Sign on user log book.


Sample loading

1. Take out uranyl acetate (4%) and glutaraldehyde (25%) from refrigerator.
2. Dilute uranyl acetate to 1% and glutaraldehyde to 2.5%.
3. Support the discharged grid using the special tweezers. Use the edge of the grids.
4. Apply 8 μL of sample solution onto the grid carefully and incubate for 10 min. (The incubation time is determined by the sample property and concentration).
5. Wick off the solution using a piece of the Whatman filter.
6. Apply 5-8 μL of 2.5% glutaraldehyde onto the grid and incubate for 1-5 min.
7. Wick off the solution with Whatman filter.
8. Apply 2×10 μL volumes of deionized water to rinse the grid.
9. Wick off water using the Whatman filter.
10. Apply 8 μL of uranyl acetate for 1 min to stain the proteins.
11. Wick off the staining soluion with Whatman filter.
12. Air-dry the grid.
13. Place the grids into the gird box.

Personal tools