Difference: PlungeFreezing (1 vs. 10)

Revision 1005 Apr 2015 - Main.AshleighRaczkowski

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Plunge freezing

Jump to Principles

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

Added:
>
>		 * CP3PlungeFreezerManual.pdf: CP3 Plunge Freezer Manual

META FILEATTACHMENT attachment="CP3PlungeFreezerManual.pdf" attr="" comment="CP3 Plunge Freezer Manual" date="1428244039" name="CP3PlungeFreezerManual.pdf" path="CP3PlungeFreezerManual.pdf" size="1443149" stream="CP3PlungeFreezerManual.pdf" user="Main.AshleighRaczkowski" version="1"

Revision 922 Oct 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Plunge freezing

Changed:
<
<

Jump to Principles

>
>

Jump to Principles

 

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

Revision 822 Oct 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Plunge freezing

Changed:
<
<
>
>

Jump to Principles

Added:
>
>
 

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

Revision 721 Oct 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM
Changed:
<
<

Plunge freezing

>
>

Principles & Protocols

Added:
>
>

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

Revision 601 Sep 2009 - Main.KdDerr

Changed:
<
<
META TOPICPARENT name="SamplePreparation"
>
>
META TOPICPARENT name="CryoSP"
 Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

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

Revision 526 Aug 2009 - Main.KdDerr

 
META TOPICPARENT name="SamplePreparation"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Plunge freezing

Deleted:
<
<		Contents
 

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

Revision 409 Jun 2006 - Main.KdDerr

 
META TOPICPARENT name="SamplePreparation"
Changed:
<
<		Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Plunge freezing

Contents

You will need:

  • Several pairs of tweezers
  • Ethane
  • LN2
  • Quantifoil R2/2 100 holey carbon film 200 mesh Cu
  • Humidity tent
  • 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

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

Plunge freezing

Contents

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

Deleted:
<
<

Revision 322 Mar 2005 - Main.DavidStokes

 
META TOPICPARENT name="SamplePreparation"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Plunge freezing

Contents

You will need:

  • Several pairs of tweezers
  • Ethane
  • LN2
  • Quantifoil R2/2 100 holey carbon film 200 mesh Cu
  • Humidity tent
  • 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

Added:
>
>

Revision 218 Mar 2005 - Main.DavidStokes

Changed:
<
<
META TOPICPARENT name="CryoEM"
>
>
META TOPICPARENT name="SamplePreparation"
 Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Plunge freezing

Contents

You will need:

  • Several pairs of tweezers
  • Ethane
  • LN2
  • Quantifoil R2/2 100 holey carbon film 200 mesh Cu
  • Humidity tent
  • 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

Changed:
<
<		-- DavidStokes - 18 Mar 2005
>
>		-- DavidStokes - 18 Mar 2005
Deleted:
<
<

Revision 118 Mar 2005 - Main.DavidStokes

 
META TOPICPARENT name="CryoEM"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Plunge freezing

Contents

You will need:

  • Several pairs of tweezers
  • Ethane
  • LN2
  • Quantifoil R2/2 100 holey carbon film 200 mesh Cu
  • Humidity tent
  • 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

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