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THIS PROTOCOL INCLUDES THE USE OF DANGEROUS MATERIALS AND INSTRUMENTS. THE PROTOCOL IS FOR TRAINED USERS ONLY.

Startup Checklist

  • Check tools and materials
  • Check Vitrobot log for problems
  • Check Vitrobot for problems
  • Check ethane tank for problems

 

Wrap-up Checklist

 

  • Removed blotting paper from Vitrobot
  • Leave Vitrobot door ajar (allows chamber to dry out)
  • Put Vitrobot tweezers, blotting paper, and grids away
  • Remove humidifier and fully empty water
  • Turn Vitrobot off
  • Check ethane tank
    • Empty excess ethane in pressure valve
    • Close all valves
  • Put cryo-grids in long term storage
  • Put away all tools and materials
  • Clean up area

 

Main Vitrobot Protocol

 

Startup –

  1. Glow discharge grids
  2. Turn Vitrobot on
  3. Attach blotting paper to Vitrobot blotting pads
  4. Attach Vitrobot humidifier and fill w/ H2O
  5. Set Vitrobot humidity and temperature (typically 4 oC and 100%, ~30m)
  6. Setup and organize working space
  7. Plan blot parameters (blot time, sample quantity, etc.)
  8. Fill ethane pot w/ LN2 and let cool
  9. Dispense ethane
  10. Place grid box in ethane pot LN2 position

 

Grid Freezing –

  1. Set Vitrobot blotting settings (blot time, etc.)
  2. Pick up grid w/ Vitrobot tweezers and attach tweezers to Vitrobot
  3. Raise tweezers
  4. Place ethane pot into plunge freezing position on Vitrobot
  5. Pipette sample onto grid
  6. Wait if desired (typically 1m)
  7. Blot and plunge freeze grid
  8. Top off ethane pot LN2
  9. Detach Vitrobot tweezers from Vitrobot
  10. Move LN2/ethane to comfortable place
  11. Wait for fog to clear above LN2 in ethane pot
  12. Quickly transfer grid to ethane pot LN2
  13. Place grid into grid box while keeping it under LN2
  14. (Move a full grid box to falcon tube LN2 transport dewar)
  15. Repeat grid freezing until finished

 

 

 

Protocol Details

 

  1. The time between grid glow discharging and grid preparation will affect the surface chemistry of the grid. Immediately after glow discharge highly reactive groups exist on the carbon (oxygen radicals and other unstable groups). The longer the grids sits in atmosphere the more reactive groups will change to less reactive groups (ethers, carbonyls, etc.). After months to years the grid will be hydrophobic again.
  2. The switch is located on the back right bottom of the Vitrobot
  3. Use the white rings to hold the blotting paper in place. Be careful not to damage the blotting pads or touch the paper without gloves. One can make their own blotting papers if they need special blotting paper (e.g., ash free which has no Ca2+)
  4. The humidifier is attached by sliding it up under the main chamber and then rotating it a bit to bring the dot to the front and lock it in place. Be careful to make sure the red O-ring is in place and does not get crimped. Use the syringe to add 50 mL of miliQ H2 Just after adding the water, when the syringe is still attached to the insertion tube, fully draw out the plunger and then push it back it pushing air through the line. The water should last 3-4 hrs.
  5. Set the temperature first. The humidifier will not start until the temperature gets close to the setting you want.
  6. Arrange your workspace. Since plunge freezing tools quickly become iced up time is important. You don’t want to waste any time messing with things after you fill the ethane pot. With that in mind, layout where your grids will be, where the tweezers are, where your kimi wipes are, fill your LN2 transport dewar, etc.
  7. Write out your blotting parameters so you don’t have to make a rushed decision later or forget.
  8. Fill the ethane pot with LN2 and cool it and the spider. Keep the pot covered with a kimi wipe when possible to reduce ice buildup.
  9. Safety – Ethane is very dangerous. Wear safety glasses. Ethane, unlike LN2, freezes on contact and a small drop can blind you permanently. In addition, ethane freezes at LN2 temperature and with this setup the surface of the ethane can freeze. If that happens ethane can spurt up and into your face should the frozen surface be punctured. and keep heat sources away (such as blow dryers). If the spider is stuck to the ethane cup it can be dangerous to try prying it off using force. One can place a nickel on top of the spider (it fits perfectly) for a few seconds to melt the frozen ethane that causes the Spider to become stuck to the ethane cup. This is safer than trying to pry it off and risking a liquid ethane splash.
    1. Once everything is at LN2 temperature you can add ethane to the bronze ethane cup.
    2. Open the ethane tank main valve a crack to let ethane into the pressure valve.
    3. While moving the ethane hose tip around in the bottom of the ethane cup, very slowly open the pressure valve on the ethane tank until you get a slow fog of ethane emerging.
    4. Continue moving the tip around the bottom of the cup until a layer of liquid ethane has built up.
    5. Ethane can freeze and clog the tip. If this happens, close the pressure valve, remove the tip from the cup, and let it warm up while pointed away from you. When the blockage has melted you can resume as before.
    6. Once a layer of liquid has built up you can stop moving the tip around and let the ethane fill.
    7. As more ethane fills the cup you can increase the flow rate, but be careful not to open the pressure valve past 5 PSI as that can blow the liquid ethane everywhere like a can of compressed air.
    8. Fill the ethane cup to 2 mm below the top of the cup.
    9. Before plunge freezing, watch for the ethane to freeze on the sides and bottom of the ethane cup. You won’t get vitreous Ice if the ethane is not below ~-165 o The frozen ethane is a key indicator that you will get vitreous ice.
    10. If the ethane freezes up too much you can condense a bit more ethane to melt it or melt it using something metal like tweezers or a warmed up spider.
  10. Place 1 grid box in the grid box indentation in the ethane pot. Do not place more than one even if you plan to make more than 4 grids as it will only lead to excess ice contamination.
  11. Go to the options page on the Vitrobot. Set your blotting time, and any other parameters you prefer. Blot force should remain unchanged unless you specifically need a specific setting. Should you change blot force, return it to the previous setting when you are done.
  12. Pick up grid and place attach tweezers to plunging rod.
    1. Pick up a grid with the Vitrobot tweezers and lock it in place with by moving the black band down (stop at the 1st ridge). Test the hold on the grid by taping the tweezers against your finger with the grid oriented parallel to the taping direction. If the grid stays in place it should hold during plunging.
    2. Lower the plunging rod by selecting the ‘place grid’ button.
    3. With the grid facing the sample port, attach the tweezers to the rod.
  13. Raise the tweezers.
  14. Remove the spider from the ethane pot and temporarily store in LN2 to keep it cold. Then place the pot onto the Vitrobot and raise the pot into place.
  15. Quickly, pipette your sample onto the grid.
  16. Most people wait a consistent 1 minute with the sample on the grid. However, this is not truly necessary if you can keep the time between pipetting and blotting consistent. Nevertheless, do whatever works with your sample.
  17. Select the ‘PROCESS’ button to plunge freeze your grid.
  18. Top off the LN2 in the ethane pot as doing so later will require laboratory acrobatics. Do not overfill or you will freeze your fingers moving the pot.
  19. Use both hands to carefully detach the tweezers from the plunge freezing rod. One can brace one hand on the edge of the ethane pot to help minimize tweezer movement and the possibility of smashing one’s grid.
  20. Move the ethane pot to a nearby place where it is easy to work.
  21. Moving the grid through the fog leads to ice contamination on the grid. Wait for it to settle down. This can be helped by slightly lifting the air curtain and generally being patient. As long as your grid is submerged in the liquid ethane it is safe from ice contamination.
  22. Quickly, move the grid into the LN2 outside the brass cup. It is important to do this with the grid parallel to the direction of motion, not to raise the grid into the fog layer, and to keep the grid under the LN2 once there.
  23. Move the grid through he LN2 and place it into the grid box. Keep it out of the vapor as much as possible to avoid ice contamination.
  24. Once your grid box is full, screw closed the grid box moderately tightly and move it into the falcon tube. When doing the transfer, be sure to hold the transfer dewar as close as possible to the ethane pot and transfer the grid box as quickly as possible.

Tools and Materials

 

  • Vitrobot
  • Ethane
  • LN2 (~4L)
  • Vitrobot Tweezers
  • Grid tweezers
  • Large forceps (~12”)
  • Medium forceps (~6”)
  • Grid boxes (for cryo grids)
  • Falcon tube (for grid boxes)
  • 4 LN2 dewar
  • LN2 transport dewar
  • Blotting paper (for Vitrobot)
  • Humidity gauge
  • Notebook
  • Screw driver (for grid box)
  • Safety glasses
  • Cryo gloves
  • Grids
  • Petri plate (for glow discharged grids)
  • Kim wipes
  • Paper towels
  • Lab tape (for labels)

Vitrobot Parts Identification