BECM

Leica GP2 (SOP)

Leica GP2 (SOP)

Before using the plunger, any new user has to undergo a standard training. The goal of this training is for the user to become independent enough to do the grid plunging by themselves. The facility staff will decide at which moment this happens. Once independent, the user can work in out of business hours without observation. However, every user is free to ask at any time for help and advice from the facility staff.

The plunger needs to be booked through the booking system. And if no one wants to use the machine directly after you, you have to run a bake out cycle which will last at least 70 minutes.

Leica GP2
Figure 1: Leica GP2 in sleep mode.

 

GP2_Operating_manual.pdf

GP2 operating manual by Leica

plunger preparation

  1. Touch the screen to initialize the plunger (Figure 1).
Touch screen to initialize
Figure 1: Touch screen to initialize the plunger
  1.  After initialization a warning appears to inform you about the preparation steps upcoming (Figure 2).
Replace filter paper notice
Figure 2: Prepare notice
  1. Reinsert the drip tray on the right side of the plunger (Figure 3).
drip tray
Figure 3: Inserting the drip tray.
  1. Replace the filter in the plunging chamber and attach it with the magnetic disc (Figure 4&5). Important: The machine is only calibrated for Whatman 1 paper.
pick up filter and disc
Figure 4: Picking up a filter paper and the magnetic disc.

 

insert filter in plunger
Figure 5: Inserting the filter paper into the Leica plunger.
  1. Attach the water reservoir on the right side of the plunger (Figure 6) and tighten it with the screw (Figure 7).
Reattaching the water reservoir
Figure 6: Attaching the water reservoir.
Fixing water reservoir with screw
Figure 7: Fixing the water reservoir with the screw.
  1.  Add 70 ml MilliQ water through the 3 way valve (Figure 8).
Refilling the humidifier
Figure 8: Refilling the humidifier with MilliQ water.
  1. Add the black ethane container in the cooling area (Figure 9).
  2. Add the grid box storage container into the cooling area (Figure 9).
  3. Cool and fill the area with LN2 until  just above the metal mesh (Figure 9). You will need around 1.8 l.
  4. Click 'OK' on the touchscreen (Figure 2)
LN2 refilled
Figure 9: Filled and cool LN2 container.

 

  1. To condense the ethane attach the secondary cryogen liquefier onto the ethane container (Figure 10).
ethane liquefier
Figure 10: Ethane liquefier on top of the ethane reservoir.
  1. Connect the hose with the connector to the ethane gas tank (Figure 11).
ethane hose connection
Figure 11: Connection of the ethane to the liquefier hose.
  1. Open the main valve of the ethane tank.
  2. Open the regulator valve of the ethane tank until you see gas entering the liquefier.
  3. Ethane will condense in the chamber and you can see the ethane level due to surface reflections (Figure 12).
ethane reflection
Figure 12: Reflection of the ethane during condensation.
  1. Once the ethane container is filled, close the regulator valve and the main valve of the ethane bottle.
  2. Slowly lift up the liquefier and make sure to not drop excess ethane around.
  3. Important: Make sure that the outer part of the cryo area can still move down and is not frozen in place
  4. Top up the liquid nitrogen until the mesh is just covered.
  5. Note: Make sure the ethane is cooled before starting the plunging. At this point you may want to do the glow discharging of your grid.

Front side blotting

We have two stored programs for front side blotting. One at 6 degrees Celsius and one at 20 degrees. Here the sample blotted on the same side as it was applied.

The GP2 is only calibrated for Whatman 1 paper and per design expects 4 ul of sample due to the setup of the blotting sensor. 3 ul sample seems to work always as well. 2 ul sample needs manual intervention in 50% of the cases, but can be done.

  1. In the main display you can see the chamber temperature, relative humidity, filling level of LN2 and available blotting positions (Figure 1)
  2. If you have changed the blotting paper click on the two circling arrows next to 'blot' to reset the counter to 10 (Figure 1).
main window
Figure 1: Main window of the control unit.
  1. On the main display click on 'Programs'  and select the program (either 1 or 2) called "frontside ..." (Figure 2)
Select the plunging program
Figure 2: Selection of the blotting program
  1. Note: Give the chamber enough time to cool to 6 degrees (or heat) to reach equilibrium, before starting with your first grid.
  2. On the home screen (Figure 3) you have the chance to change some parameters of your grid plunging procedure.
home screen
Figure 3: Home screen
  1. By clicking on the pencil icon next to chamber on the home screen (Figure 3, top left), you can open the display for the environment (Figure 4) to adapt the chamber temperature and the humidity.
  2. For this click on 'Set' for either option (Figure 4) and type in the desired temperature (Figure 5) or humidity (Figure 6).
  3. Confirm by clicking 'OK' (Figure 5/6)
  4. Either go back to home screen by clicking 'Main' or to the 'load specimen' screen by clicking that button (Figure 4).
  5. Note: changing the humidity is not recommended
  6. Note 2: changing the temperature will required equilibration time.
Environment screen
Figure 4: Environment settings
chamber temperature
Figure 5: Adjusting chamber temperature
chamber humidity
Figure 6: Adjusting chamber humidity
  1. By clicking on the pencil icon next to load specimen on the home screen (Figure 3), you can open the display for specimen loading (Figure 7) to adapt incubation time before blotting.
  2. For this click on 'Set' next to delay time and adjust the time (Figure 7&8)
  3. Confirm with 'OK' (Figure 8)
  4. Either go back to home screen by clicking 'Main' or to the 'Blot' screen by clicking that button (Figure 4).
load specimen window
Figure 7: Load specimen display.
pre blotting time selection
Figure 8: Adjusting the pre-blotting time
  1. By clicking on the pencil icon next to 'Blot' on the home screen (Figure 3), you can open the display for Blotting (Figure 9) to adapt blotting time.
  2. For this click on 'Set' next to Blot time and adjust the time (Figure 10)
  3. Confirm with 'OK' (Figure 10)
  4. Either go back to home screen by clicking 'Main' or to the 'Plunge/Transfer' screen by clicking that button (Figure 4).
Blot window
Figure 9: Blot display window
blot time
Figure 10: Adjusting blotting time
  1. By clicking on the pencil icon next to 'Plunge' or 'Transfer' on the home screen (Figure 3), you can open the display for Plunge/Transfer (Figure 11) to adapt the post blot delay.
  2. For this click on 'Set' next to Post-blotting time and adjust the time (Figure 11&12)
  3. Confirm with 'OK' (Figure 12)
  4. Go back to home screen by clicking 'Main' (Figure 4).
Plunge/Transfer screen
Figure 11: Plunge/Transfer screen
post-blot time
Figure 12: Adjusting post-blot time
  1. On the main display click on 'Load forceps' (or use the foot pedal). The plunging rod will move (Figure 13).
home screen
Figure 13: Home screen
  1. Pick up a grid with the plunging tweezer and lock it with the black sleeve. Ensure proper orientation.
  2. Important: The picture below shows the top side of the grid. When the 'H' on the tweezer is facing you, the top side for sample application needs to face to the left!
Loading tweezer
Figure 14: Tweezer showing the top side of the grid
  1. Load the tweezer onto the gantry of the plunger (Figure 15).
  2. Note: The 'H' symbol of the tweezer will point towards you
tweezer on gantry
Figure 15: Tweezer on the gantry
  1.  Push the foot pedal to lower the chamber and lift the tweezer.
  2. Note: The tweezer will automatically rotate, so that the top side will be facing to the right.
  3. Apply you sample through the access hole on the right (Figure 16).
Applying sample through the access hole
Figure 16: Applying the sample to the grid.
  1. Push the foot pedal.
  2. The tweezer will turn automatically and the plunging process will commence.
  3. Error Note: If you use less than 3 ul of sample, the blotting sensor may not detect the blotting. This results in a warning (Figure 17). Click 'OK' and push immediately the 'Plunge' button. Grid can still be used!
blot warning
Figure 17: Possible blotting error
  1. After plunging the tweezer can be removed from the gantry, while staying in the cold area
plunged grid
Figure 18: A grid plunged in ethane.
  1. Hold the grid with the tweezer and loosen the black sleeve.
  2. Optional: Touch your grid on a filter paper that you placed in the LN2 chamber to wick away excess ethane (Figure 18).
  3. Transfer your grid into the grid box.
  4. Push the foot pedal to lift the gantry (Figure 19).
Plunger ready for more grids
Figure 19: Gantry in the upper transfer position
  1. Repeat for the other grids while varying the plunging time.
  2. Note: The next foot pedal push will move the gantry into the tweezer loading position.
  3. After four grids are plunged and added to your grid box, close the grid box
  4. Make sure the is still LN2 in the Cryo transfer container.
  5. Put the lid onto the cryo transfer container (Figure 20).
Putting the lid on the cryo transfer container
Figure 20: Putting lid on cryo transfer container
  1. Screw the M4 cryotool to the transfer container (Figure 21).
picking up cryo transfer container
Figure 21: Picking up cryo transfer container
  1. Move your grid box with the cryo transfer container into a styrofoam Dewar for transfer into the grid storage system.
  2. Once finished with your sample, continue with the bake out procedure.

Back side blotting

  1. Select the program

Hanging drop blotting

  1. Select the program

Multiple Application

  1. Select the program

Bake out cycle

The bake out should be run after every use and takes around 70 minutes to complete

  1. In the main window click on 'Settings' (bottom left, Figure 1).
main window
Figure 1:Main window panel for Settings button.
  1. In the settings window, click on 'bake out' (Figure 2).
settings window
Figure 2: Settings window with bake out button.
  1. A warning message appears (Figure 3).
  2. Remove the ethane vessel and the cryo transfer container (if not done yet during grid transfer) The plunging area should look like Figure 4.
remove vessel warning
Figure 3: Remove cryogen warning.
plunging area for bake out
Figure 4: Plunging area ready for bake out
  1.  A new warning appears (Figure 5)
  2. Remove the filter paper and the magnetic disc. The chamber should  look as in Figure 6.
filter paper removal warning
Figure 5: Filter paper warning
chamber with filter paper removed
Figure 6: Blotting chamber ready for bake out.
  1. Another warning appears (Figure 7). Note: We do not have a viewing system.
  2. Open the front door of the blotting chamber as in Figure 8. 
viewing chamber open warning
Figure 7: Warning to open chamber door
open blotting chamber door
Figure 8: Leica with open chamber door.
  1. On the Panel click Start to begin the bake out (Figure 9).
  2. The timer will only start once the liquid nitrogen is evaporated and the temperature reached 100 degrees (Figure 10). 
Settings window
Figure 9: Bake out window with Start button.
bake out running
Figure 10: Bake out is running.
  1. Remove the humidifier reservoir by loosening the screw (Figure 11) and carefully moving down with the container (Figure 12)
  2. Remove the excess liquid.
  3. Dry the chamber with paper.
unscrewing the humidifier reservoir
Figure 11: Loosen the screw of the humidifier reservoir
pulling out the humidifier reservoir
Figure 12: Pulling out the humidifier reservoir.
  1. Remove the dripping tray (Figure 13).
  2. Remove access liquid.
  3. Dry the dripping tray with paper.
removing dripping tray
Figure 13: Remove the dripping tray
  1. Wipe off access liquid from the nozzles at the humidifier reservoir with paper (Figure 13)
  2. Remove big puddles of water in the blotting chamber with paper.
  3. Put all Leica parts back into the box as shown in Figure 14.
Leica tool box
Figure 14: Leica toolbox