Cryo Specimen preparation

Prepare holey grids on filter paper (methanol soaked). Thick Carbon coat on grids. Glow discharge before use, for ~50 sec. For membrane samples, prepare very thick carbon coat on freshly cleaved mica. Float onto water, above a filter paper with grids, and drain out the water slowly to lower the film onto the grids. glow discharge 30 sec in amylamine at 2x10-1 Torr. This can be done by putting 10 µl of amylamine on a piece of filter paper in the chamber. The discharge should be blue-purple instead of pinkish-purple as it is in air. It may be best to do the glow discharge the day before the cryo grid preparation. Maybe easier to use copper-rhodium grids, because the colour difference makes it easier to recognize the carbon side (coat on rhodium) when applying the sample (especially in the dark).

Sample vitrification

Prepare ethane dewar and plunger. Test blotting time of grid with specimen on a test grid. 1 mg/ml protein seems to work, avoid high salt. Melt the ethane with copper rod, wait for it to start refreezing. Blot from the front and plunge just after the filter paper detaches from the grid - slight darkening of damp patch on the filter paper. Blotting normally takes a few seconds. Transfer grids from the ethane to N2 and keep under N2 thereafter.

Cambridge methods for membrane crystals: Humid chamber and cold room: if the plunging can be done in a humid atmosphere, the blotting time can be longer, and should be more reproducible. For membrane samples on solid carbon films glow discharged in amylamine, at 4° & in high humidity, 3.5 ul of sample was loaded on the grid for 1.5 min (while vertical), and the blotting time was 18 sec (range 10-25 sec). The filter paper (damp from storage in cold room) was pushed hard against the grid. They also have a little heater in the ethane so it is alway liquid.

Storage of cryo grids

Grids can be stored in the plastic disks, in plastic test tubes on strings (Falcon tubes with holes punched in cap) in the storage dewar. Avoid leaving grids in open containers of liquid N2 for long periods, which causes ice contamination. Ethane and N2 used in freezing and transfer should be changed after ~½ hr and dry containers used.