Leica EM GP2 Standard Operating Procedure

1. Purpose

Setting up, operating, and shutting down the Leica EM-GP2 in a safe and effective manner for the preparation of frozen cryo-EM of samples. Note: This SOP is for BSL-1 work only.

2. Definitions:

2.1 Leica GP2 is a vitrification device for freezing specimens to use for cryoEM analysis.

2.2 Liquid Nitrogen (LN2) is a cryogenic liquid stored under pressure.

2.3 Ethane is classified as a flammable gas.

3. Supplies & Equipment 

  • PPE (BSL-1)*(also see site specific instructions)
    • Laboratory Coat
    • Nitrile Gloves
    • Goggles / Safety Glasses
    • Cryogenic Gloves
    • Face Shield
  • Chemicals/Reagents
    • Liquid Nitrogen
    • Ethane
    • 70% Ethanol
    • Distilled Water
    • Sample
  • Filter Paper (Pre-punched Whatman #1 or #541)
  • Pipette and Tips
  • Tweezers for misc. grid handling
  • Leica GP2 Tweezers
  • Glow Discharged / Plasma Cleaned EM Grids
  • Grid Storage Boxes/Buttons
  • Grid Box Lid Opening Tool (Screwdriver or TFS Pin Tool)
  • LN2 Transfer Dewars
  • Forceps (fine tip and long tweezers)

4. Procedure

4.1 Instrument setup and cryogen preparation (takes 30-60 min) *(also see site specific instructions for 4.1)

Once you have added the 60 mL the touch screen warning will turn off. Turn the full/drain hose connection to closed and hang it on it’s storage clip.

This will take ~1.8 L of LN2. Refill as needed during the freezing session. The LN2 level should remain below the metal mesh while the cryogen and transfer containers remain cold in a nitrogen vapor environment.

Typical settings:

  • Temperature 4-25°C
  • Relative Humidity 0-99%
  • Cryogen Temperature -175°C
  • Cryogen GN2 flow 100%

Note: humidifier will only run when chamber temperature

Typical blot times are 0.5-3.0 s.

Adjust Horizontal and Vertical blot positions: load a blank grid on the tweezers then load the
tweezers onto the plunge rod. Next, click the “Set” button. The chamber will lower to blotting
position and you can adjust the tweezer position using the control panel. You can use the
binoculars if available. *(see site specific details for typical settings).

4.2 Freezing Cycle / Plunge Freezing

Replace blot papers after every 10 blots or every 20 minutes, whichever occurs first.

Optional: Reset the Leica GP2’s counter so the system will keep track for you

Please note, the tweezers can only be attached to the plunge rod in one orientation. Be mindful of which side your foil faces, so that you can apply your sample to the side you want (the system allows you to rotate the tweezers during the plunging protocol if incorrect).

If you did not enable automatic plunging, the “PLUNGE” button will need to be pressed when it becomes available. If you did set up automatic plunging, the system will plunge the grid into liquid ethane after blotting.

The grid box is in the silver transfer container and you will need to quickly move between the ethane cup and transfer container.