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Principles & Protocols

Protocols - Carbon Sandwich

Jump to Principles

You will need:

• Gloves
• Parafilm
• Tweezers
• Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
• Carbon Coated Grid
• Carbon Coated Mica
• Pipette
• Filter paper
• Small weight boat

Procedure:

• Glow Discharge carbon coated grids
• Put on gloves!
  • Most negative stains are very toxic, especially Uranium salts.
• Place about 3ml of the stain in the weight boat
• Cut a small piece of the carbon coated mica (~2x2mm), keeping track of the carbon side
• float the carbon film on the negative stain
• Secure the grid in a pair of tweezers
• Pipette sample (~3 microliters) onto glow-discharged grid.
• After about 30 seconds, blot the excess sample from the grid with filter paper
• Dip the grid on the boat with the negative stain and pick up the floating carbon film with the grid, with the grid having the carbon side pointing upwards
• Blot the grid from the back side
• Place the grid on a piece of filter paper and let it sit for a few hours

Possible variations:

• This method works nicely with Uranyl formate, but in principle any stain should be fine.
• Trehalose or other materials can be used to produce some degree of embedding and better preservation.
• The method has been reported in much more complicated ways, so it seems to be limited only by the experimenter's imagination.

-- RubenDiaz Sep 4 2007