Hazards:

-High Pressure and Combustible Ethane, Burn Hazard

Caution is required when:

-Handling delicate grids.

-Transferring liquid nitrogen (N2).

-Using fine-tipped precision tweezers.

***Do not access the instruments without training and authorization from Collazo. You CAN NOT be trained by another lab member. No secondary training.

***This machine uses fast moving components to quickly plunge EM grids into liquid ethane. If protocol is not followed correctly, serious damage can be done to the machine and the operator.

Preparing the cooling apparatus

  1. Wear safety glasses MANDATORY. You must have the proper PPE to proceed. Wear safety glasses, a lab coat, and gloves.
  2. Obtain a dewar of LN2 (~3L).
  3. Ensure all components/tools are free of moisture.
  4. Fill the outer chamber of the gray styrofoam container to the brim with LN2.
  5. Place the brass cooling vessel in the center of the gray styrofoam container. Fill the brass vessel with LN2 and allow evaporation to occur until no more LN2 is observed in the vessel. Keep the outer chamber (Styrofoam portion) full of LN2.
  6. Place the Spider over the brass cooling vessel.
  7. Close the valve on the ethane tank by twisting the outward valve (red/left-side) in the decrease direction (left) until completely closed. You will not feel a stop. Do this for 4-5 full rotations. If you feel the handle coming loose, do not proceed.
    1. Note: This is opposite most screw-like mechanisms.
  8. Slowly open the inward valve (right-side) until the pressure jumps; this is usually around 600 psi.
  9. Place the tip of the liquid ethane hose on the bottom of the dry brass cooling vessel.
  10. Very slowly open the outward valve (red/left-side) by twisting in the increase direction (right) until a slight hiss is heard.
    1. The gauge should not visibly move but remain between 0 and 1.
  11. Using the tip at the end of the hose, aim the ethane at the chilled brass cooling vessel and move the tip of the hose around the surface of the brass vessel as if applying a thin coat.
    1. As this happens, ethane(g) will cool and condense to ethane(l).
  12. Fill the brass vessel until it is at least ¾ full with condensed ethane(l). Do not overfill. Ethane reacts violently on contact with LN2. Remove the tip before the vessel is full.
  13. Shut off the outward valve first by twisting in the decrease position, then close the inward valve completely.
  14. Carefully fill the Styrofoam plunging vessel with LN2 (avoiding the ethane).
  15. Wait until the ethane becomes opaque in appearance (but not frozen white) and remove the spider.
  16. Place slotted grid boxes in the small circular indentions of the container; use as many as is necessary to prepare all of your grids. Ensure that the LN2 level does not fall below the grid box level by adding LN2 as needed. 

Loading the blot paper

  1. Attach a piece of pre-cut blot paper to the blotting mechanism.
  2. Place the plastic rings over the paper to ensure attachment to the mechanism.

Preparing the grid

  1. Using the Vitrobot compatible fine-tipped precision tweezers, grab grid and lock the tweezers in place.
    1. Be careful when using these tweezers; NEVER try to pick up the grid directly from a hard surface as this will blunt the tip making them useless.
  2. Turn on the Vitrobot (switch is in the back); select [Reset Blotpapers]. Each blot paper can be reset 15 times (16 grids).
  3. Using a stylus, select the desired parameters under the [Options]
    1. The number of blots, duration of each blot, and time between blots can all be adjusted and depends on the type of sample to be loaded.
  4. Select [Console] to return to the previous screen.
  5. Select [Place New]; the loading dock for the tweezers will descend.
  6. Using both hands, place the tweezers at the mount and slide it into place.
    1. When correctly attached, a slight “click” can be heard as the ball bearing locks into place.
    2. It helps to approach the mount at a 45° angle, placing a finger on the rod to firmly click the tweezers in place.
  7. Carefully place the entire Styrofoam apparatus on the mount of the Vitrobot.
  8. DO NOT PROCEED IF YOU HAVE NOT REMOVED THE SPIDER.
  9. Select [Continue] and tweezers will ascend into the blotting chamber. Select [Place Ethane Container]. The apparatus will move backward until directly under the tweezers. The container will rise to be flush with the bottom of the blotting chamber. Select [Start Process]. The tweezers will descend slightly in the blotting chamber to allow the grid to be aligned with the red sample loading ports on the side of the chamber.

Loading a sample

  1. Using a micropipette, dispense 1-3 uL sample on to one or both sides of the grid (as sample requires).
    1. Slide the red seals over to access the loading chamber, then close them after loading.
    2. Be careful not to touch the tip of the pipette directly to the grid or risk damage; use the adhesive properties of the solution to transfer the droplet.
  2. Select [Continue] and the machine will begin the blotting process then immediately afterward plunge the grid directly into the ethane below. The apparatus will descend along with the tweezers.
  3. Once finished, use both hands to carefully remove the tweezers from the mount.
    1. It is essential that the grid remain submerged in ethane the entire time; move slowly if necessary to avoid removing from ethane or bumping the grid against the sides.
  4. As quickly and precisely as possible, transfer the grid from the ethane in the LN2 bath at the side of the vessel; speed is important or exposure to moisture will result in ice contamination.
  5. After about 5 sec, transfer the grid to the grid box.
    1. Be careful not to transfer too quickly or with excessive force or the grid will bend.
  6. Select [Continue] and [Place New Grid] if more than one grid is being prepared.
  7. Repeat steps 6-9 of the Preparing the grid portion and steps 1-6 of the Loading a sample protocol for each grid. Periodically replenish the LN2 level while avoiding contacting the ethane.
  8. Once all grids have been prepared, select [Exit] AND turn off the machine using the button in the back.
  9. Remove used blotting paper.
  10. Place the grid holders in the appropriate storage.
  11. Leave LN2 and ethane to evaporate in a secure location.

Suggested parameters (courtesy of Paul Seidler): 

Program for peptide xtals:

Blot force = 16 (-25 to 25)
Blot time = 18-24 sec
wait time = 0 sec
humidity = 0

Program for fibrils:

Blot force = 1
Blot time = 7 sec
wait time = 2 sec
humidity = 100