Grid Clipping Standard Operating Procedure

1. Purpose

Successfully clip grids into TFS autogrid rings.

2. Definitions:

2.1 An autoloader system is a robotic system for loading cryo-EM grids into the microscope, in which the user inserts clipped grids into a cassette that is automatically loaded into the microscope using a retractable arm.
2.2 C-clips and rings are attached to a cryo-EM grid to allow a robotic retractable arm to grip and load the grid in an autoloader system.

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

2.4 Definition of terms for tools/equipment can be found in Figure 1.

3. Supplies & Equipment 

  • PPE (BSL-1)
    • Laboratory Coat
    • Nitrile Gloves
    • Goggles / Safety Glasses
    • Cryogenic Gloves
    • Face Mask
  • Table-top clipping station with aluminum & brass transfer pedestal
  • C-clips (one per grid)
  • Autogrid rings (one per grid)
  • Autogrid tweezer
  • Fine tip tweezer
  • Autogrid storage boxes and lids
  • Autogrid box lid gripper tool
  • C-clip clipping tool (one per grid)
  • EM grids with your vitrified sample in a grid box under LN2.
  • Sample transfer dewar
  • Liquid nitrogen storage dewar
  • Screwdriver (if needed for screwed grid boxes)
  • Large forceps
  • Hair dryer or heat block (if needed to dry tools)

4. Procedure

4.1 Loading the C-clips into the clipping tool

  1. C-clips should be circular and not bent or distorted
  2. Do not re-use recovered C-clips or rings!

If you need to re-use a cold C-clip tool, be especially sure to thoroughly warm it before using it again or else it may get stuck when you try and eject the C-clip. This can be done with a hair dryer or heat block.

4.2 Preparing the Clipping Station

During clipping, the LN2 should be at the upper lip of the aluminum portion of the transfer pedestal so that you clip in the N2 vapor layer. You can also clip with the LN2 level above the aluminum such that you clip under liquid nitrogen if contamination from humidity is an issue, but this increases the chance of damaging the grid if liquid nitrogen is forcibly displaced during the clip ejection step.

  1. Using a tweezer, add C rings to the brass pedestal to cool
  2. Place the prepared clipping tools along the notched area in the transfer station
  3. Transfer your cryo grid box with unclipped grids to the well of the aluminum pedestal
  4. Cool down a screwdriver and loosen the lid of the grid box
  5. Place a new autogrid storage box with pin type lid in the well of the aluminum pedestal
  6. Cool down a gripper tool and remove the lid of the pin type lid of the new grid box. You can screw the pin type lid into the pedestal between the grid boxes to secure them during clipping.

4.3 Clipping grids

Make sure that the “lip” on the ring is facing upwards, using magnification if necessary

  1. The C-clip should be lying flat and the grid should be flush.
  2. Do not load if grid is torn or sticking up above the rim of the ring.
  3. Improperly clipped grids can result in instrument downtime and therefore cannot be loaded onto the microscope.

4.4 Cleaning up

5) Chemicals:

    1. Liquid Nitrogen

6) Waste Disposal:

    1. Follow facility procedure for proper disposal.