1) Log into the toolsquare unit with your badge to switch on electricity toward the system.

2) If the electricity of the CP3 does not turn on immediately, switch on the Cp-3 with the toggle switch on the right side

3) Open the valve for the pressurised air (up position). The gauge will indicate around 5 bar.

4) Pour liquid nitrogen in the liquid nitrogen workstation until the height of the metal mesh and wait until liquid nitrogen temperature is reached.

5) Condensate ethane gas slowly into the ethane pot within the liquid nitrogen workstation. Important: Wear safety goggles to protect yourself against spillage in your eyes.

6) Place an empty cryo grid box in the workstation. Set the ethane temperature controller to -176 degrees. Add more liquid nitrogen to cool down the system again.

7) Glow discharge your grids

8) Switch on humidifier. The humidifier will light up when in use.

9) Change the blotting paper. Note: You can also use one blotter on one side and the blank on the other side to do single sided blotting.

a) remove one plunger and replace it with the dummy (here visible on the left).

b) remove the old blotting paper, plastic and pin with a flat tweezer from the blotter

c) put the metal pin in the center of the assembly station

d) add a fresh piece of Whatman paper. Important: Different types of Whatman paper will affect the blotting behaviour.

e) add the plastic spacer to the assembly.

f) finish the assembly by pushing the blotter down on the pin.

g) control the Gentle blot position on the blotter. It affects the distance between the blotter and your grid and thereby the blotting force.

h) Remove the dummy blotter from the CP-3 and insert the freshly assembled blotter back. Push it in completely! Important: Ensure that the red o-ring is no more visible. Otherwise the blotting will not work.

i) Repeat steps a-h for the right blotter

10) Pick up the freshly glow discharge grid with the Cp-3 tweezer. Important: This tweezer has a very fine tip. Handle with care.

11) Keep the blue button on the plunging rod pushed to open the lock mechanism. Insert the tweezer in the plunging rod until it is completely in. Make sure that your grid does not touch the ethane container, so it does not bend. Release the blue button and confirm that the tweezer is locked in position before releasing the tweezer. Important: Ensure that the tweezer is firmly attached as it will otherwise fall off and break; this is a costly part.

12) Lift up the plunging rod. You will hear a click sound once it is locked in its top position.

13) Hit reset button on the CP-3 and carefully close the safety shield.

14) Apply 3 ul of sample onto your grid. If you have a support layer e.g. thin carbon or Graphene oxide your sample needs to incubate for 30 s. Hint: You can do steps 15-18 in the meanwhile.

15) Rotate the plunging rod by 90 degrees.

16) Rotate the blotting papers by 90 degrees.

17) Set the blotting time.

18) Switch of humidifier. Note: Switching the humidifier on and off between multiple grid freezings in optional;

19) Hit the start button on the CP-3. The blotting will commence.

20) Open carefully the safety shield.

21) Hold with one hand the tweezer. With the other hand push the blue button on the rod lift up the plunging rod. Once the tweezer is free you can put the plunging rod down. Important: Your grid should stay in the cold ethane.

22) Regrab the tweezer below the black plastic, so that you hold the grid manually.

23) Remove the black plastic and use you right hand to hold the tweezer in a more comfortable position.

24) Quickly transfer the grid from the ethane into the nitrogen cooled air. You do not need to touch the liquid nitrogen itself. Important: Do not move up too much during this as you will pick up contaminants on your grid and might warm it up in the process leading to crystalline ice.

25) Slowly transfer your grid into the gridbox. Hint: It is enough to only go partially into the slot with the grid. Once you release the tweezer it will fall to the bottom of the grid box.

26) Repeat steps 9-26 until finished. Note: After 4 blottings you will have to change the blotting paper again. Important: Before picking up the next grid make sure that you tweezer is dry (use hair drier)

Common Problems:

P: The blotting papers did not move during the blotting.

S: Are the blotters inserted completely? The o-ring should not be visible anymore.

S2: Make sure that you pressed the Start button properly/hard enough.

P: The plunging rod did not come down.

S: The safety shield needs to be closed for that.

P: My ethane is solid

S: Increase the set temperature on the CP-3