How to use the Leica Grid Plunger GP2

Switch on the Leica Plunge freezer

After startup the plunger will show warnings to replace the filter, water and nitrogen. Confirm these warnings and continue.

Choose a program

assemble the humidifier

Fill a beaker with more than 100 ml MilliQ water.

The humidifier needs around 90 ml

Fill the syringe with 60 ml of water.

Connect the syringe and fill the resevoir.

Detach the syringe and fill it with another 30-40 ml of water.

The plunger will give a warning, when a high level of water is reached.

The saved programs contain settings for temperature and humidity, blotting time and other presets.

Typical values for single particle freezing are: 10°C and 99% of humidity. Blotting times between 3.5 to 4 seconds work well. The cryogen should be at -185°C

Single particle grids are usually blotted from the side of the support film. There is the option to add an additional move of the blotting paper, which is guided by infrared light. A standard value is an additional move of 1.4 mm to follow the liquid drop.

The carbon side of the cryo EM grid is typically oriented towards the center of the grid box.

Place the grids with the carbon side facing upwards on a glass slide.

Typical grids are Au 200/1.2/1.3 grids (number of mesh squares/diameter of holes/distance between holes)

Place the large petri dish without a lid on a small petri dish as a spacer in the chamber of the glow discharger.

follow the instructions on the glow discharger

Set it to 20 mA and glow discharge for 90 seconds

Take the metal container out of the accessory box and place it in the Plunge freezer.

Place the lid on the container.

Add the cryogen supply lid as well.

A typical cryogen is a mixture of ethane and propane gas, that is liquid at -185°C

Pour liquid nitrogen into the dewar of the plunge freezer to cool down the system.

Avoid pouring liquid nitrogen into the cryogen container

Add nitrogen until the level is reaching the grid at the bottom and until the nitrogen stops boiling.

Open the main valve first.

Open the second valve carefully and watch the cryogen container through the little window.