Vitrobot

  1. Turn on vitrobot using the switch at the back
  2. Attach the silver cylinder to the base of the chamber and fill with 60ml deionized water using the 60ml syringe
  3. Set the humidity to 100% and the temperature to 4C using the touchscreen
  4. Close the door and open the side ports, leave to equilibrate
  5. Start the glow discharge of your grids
  6. Fill the Styrofoam container, the box and the flask with liquid nitrogen
  7. Put the spider and the copper cup in the box to cool
  8. Put the grid box in the Styrofoam container
  9. Use the large tweezers to pick up the copper cup, pouring the liquid nitrogen out. Place it in the center of the Styrofoam container and put the spider on top. Use the plastic piece to evaporate any remaining nitrogen from the cup.
  10. Open the main valve of the ethane container and adjust the pressure to 5. Holding the tubing above the cup, slowly open the small valve until ethane is released. Put the tip into the bottom of the cup and move it back and forth until you hear bubbling. At this point you can slowly increase the flow and fill the cup up to the level of the bottom of the spider.
  11. Cover the Styrofoam container and wait 5-6 minutes until the ethane starts to solidify. Remove the spider, melting the ethane with the back of the tweezer if necessary to release the spider. Top up the liquid nitrogen to the level of the outer divots.
  12. Place the filter papers on the pads, and run through a cycle to push them together. Close the side vents.
  13. Pick up the edge of a grid so that the carbon faces your preferred pipetting side. Tap the tweezer against your hand to ensure the grid is held tightly.
  14. Click place new grid on the vitrobot. Attach the tweezer to the Vitrobot, it should click into place.
  15. Press continue.
  16. Place the ethane container on the vitrobot and press ‘place ethane container’
  17. Press Process
  18. Pipette 3ul of sample onto the grid, making sure the pipette does not touch the grid, and the drop spreads out.
  19. Press continue
  20. Fill the ethane container up to the level of the outer divots.
  21. Holding the tweezer steady with one hand, use the other hand to remove the tweezer from the vitrobot
  22. Rest the tweezer on the edge of the cup for stability and move the ethane container to the benchtop
  23. Remove the clamp from the tweezer
  24. Make sure the grid box is open to the desired position
  25. Quickly move the tweezer into the liquid nitrogen
  26. Move the tweezer to the grid box and place the grid inside
  27. Cover the ethane container and dry the tweezer. Repeat from step 13
  28. After ~10 minutes/ 4 grids, replace the filter papers