Principles & Protocols

Protocols - Plunge freezing

You will need:

• Several pairs of tweezers
• Ethane
• LN2
• Quantifoil R2/2 100 holey carbon film 200 mesh Cu
• Screwdriver
• Cryo grid boxes
• Filter paper cut into strips
• Wide mouth Dewar

Procedure:

• Put on goggles!
• Put on gloves!
• Raise humidity in tent to 70% or higher
  • turn on compressed air with bubblers
  • spray water on inside walls of tent
• Fill pool with liquid nitrogen
• Use ethane to blow liquid nitrogen out of specimen area
• Adjust the speed of the gas to fill pool with liquid ethane
• Keep the tip of the hose submerged in ethane until full
• Turn down ethane to remove hose, but do not turn gas off until out of pool
• Align pool and plug in heater to prevent ethane form freezing
• Test tweezers to make sure everything is lined up
• Secure grid holey carbon grid (carbon side up) in tweezers with o-ring
• Place sample on grid with pipette. Spread sample around grid with pipette tip.
• Mount tweezers in plunge mechanism
• Blot sample
  • Put filter paper in contact with sample
  • You will be able to see the grid through the paper
  • An air bubble will form obscuring the grid
  • Wait one second
• Initiate Plunge with foot pedal
• Keep foot on pedal
• Keep tweezers in ethane
• Raise plunger (can rest left hand to stabilize sample)
• Raise o-ring on tweezers keeping sample submerged in ethane
• Quickly splash grid into LN2 to remove ethane
• Move grid into box
• Top off pool with LN2
• Adjust grid box in preparation of next sample

Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup