Bitan:Electron microscopy

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Current revision (20:54, 2 December 2009) (view source)
 
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Glow discharge the grid<br>
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Glow-discharging grids<br>
1. Clean the jar.<br>
1. Clean the jar.<br>
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7. Turn on the pump unit.<br>
7. Turn on the pump unit.<br>
8. Switch the button to glow.<br>
8. Switch the button to glow.<br>
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9. Check the glow time is 60s<br>
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9. The glow time is 60s by default.<br>
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10. Click Main button.<br>
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10. Click "Main" button.<br>
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11. Release the gas carefully to keep the pressure at 60 mba<br>
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11. Release the gas carefully to keep the pressure at 60.<br>
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12. START<br>
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12. Press "START"<br>
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13. Turn the charge switch to 4-5<br>
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13. Turn the charge switch to 4-5.<br>
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14. 60s later turn the charge back to 0.<br>
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14. Sixty sec later turn the charge back to 0.<br>
15. Release the gas.<br>
15. Release the gas.<br>
16. Turn off the Main button.<br>
16. Turn off the Main button.<br>
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17. After the gas is balanced, unclip the bell jar and take out the grids.<br>
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17. After the gas is balanced, release the jar and take out the grids.<br>
18. Put the jar back.<br>
18. Put the jar back.<br>
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19. Sign on notebook.<br>
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19. Sign on user log book.<br>
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Sample loading<br>
Sample loading<br>
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1. Take UAc 4% and glutaraldehyde 25% from refrigerate. <br>
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1. Take out uranyl acetate (4%) and glutaraldehyde (25%) from refrigerator. <br>
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2. Dilute UAc to 1% and glutaraldehyde to 2.5%.<br>
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2. Dilute uranyl acetate to 1% and glutaraldehyde to 2.5%.<br>
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3. Clip the edge of discharged grid with tweezer.<br>
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3. Support the discharged grid using the special tweezers. Use the edge of the grids.<br>
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4. 8 uL of sample solution was dropped on the grid carefully and incubate for 10 min. (the incubation time determine on the sample properties and concentration.<br>
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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).<br>
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5. Remove the solution with whatman paper.<br>
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5. Wick off the solution using a piece of the Whatman filter.<br>
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6. 5 uL of 2.5% glutaraldehyde is dropped on the grid and then incubate for 10’.<br>
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6. Apply 5-8 μL of 2.5% glutaraldehyde onto the grid and incubate for 1-5 min.<br>
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7. Remove the solution with whatman paper.<br>
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7. Wick off the solution with Whatman filter.<br>
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8. 5 uL of 1% glutaraldehyde is dropped on the grid and then incubate for 10’.<br>
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8. Apply 2×10 μL volumes of deionized water to rinse the grid.<br>
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9. Remove the solution with whatman paper and air-dry the grid.<br>
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9. Wick off water using the Whatman filter.<br>
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10. Place the grid into gird-storage box.<br>
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10. Apply 8 μL of uranyl acetate for 1 min to stain the proteins.<br>
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11. Wick off the staining soluion with Whatman filter.<br>
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12. Air-dry the grid.<br>
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13. Place the grids into the gird box.<br>

Current revision

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.

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