Difference: PPCarbonSandwich (1 vs. 14)

Revision 1421 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Changed:
<
<

Protocols - Grid preparation

>
>

Protocols - Carbon Sandwich

 

Jump to Principles

Deleted:
<
<

Carbon Sandwich

 

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.

Public website mirror

-- RubenDiaz Sep 4 2007

META TOPICMOVED by="KakoliMitra" date="1234475834" from="Main.CemPPGridsCSA" to="Main.PPCarbonSandwich"

Revision 1321 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Added:
>
>

Protocols - Grid preparation

Jump to Principles

 

Carbon Sandwich

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.

Public website mirror

-- RubenDiaz Sep 4 2007

META TOPICMOVED by="KakoliMitra" date="1234475834" from="Main.CemPPGridsCSA" to="Main.PPCarbonSandwich"

Revision 1218 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtocGrids"

Principles & Protocols

Carbon Sandwich

You will need:

  • Gloves
  • Parafilm
  • Tweezers
Changed:
<
<
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
>
>
  • 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.

Public website mirror

-- RubenDiaz Sep 4 2007

META TOPICMOVED by="KakoliMitra" date="1234475834" from="Main.CemPPGridsCSA" to="Main.PPCarbonSandwich"

Revision 1119 Aug 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtocGrids"

Principles & Protocols

Changed:
<
<

Grid preparation

>
>

Carbon Sandwich

Deleted:
<
<

Protocols - Carbon Sandwich

 

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • 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.

Public website mirror

-- RubenDiaz Sep 4 2007

META TOPICMOVED by="KakoliMitra" date="1234475834" from="Main.CemPPGridsCSA" to="Main.PPCarbonSandwich"

Revision 1019 Aug 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtocGrids"
Deleted:
<
<		 NYSBC|Cryo-EM P&P: Grid preparation - Carbon Sandwich
 
Changed:
<
<		Principles & Protocols
>
>

Principles & Protocols

 
Deleted:
<
<
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? CEM Introduction Microscope Schedule NYSBC Directory Cryo-EM public website
 
Changed:
<
<
>
>

Grid preparation

Protocols - Carbon Sandwich

Deleted:
<
<		Grid preparation

Protocols - Carbon Sandwich

Contents

 

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • 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.

Public website mirror

-- RubenDiaz Sep 4 2007

META TOPICMOVED by="KakoliMitra" date="1234475834" from="Main.CemPPGridsCSA" to="Main.PPCarbonSandwich"

Revision 912 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocGrids"
NYSBC|Cryo-EM P&P: Grid preparation - Carbon Sandwich

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
Changed:
<
<
CEMfac Home NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
>
>
CEMfac Home? CEM Introduction Microscope Schedule NYSBC Directory Cryo-EM public website
 

Grid preparation

Protocols - Carbon Sandwich

Contents

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • 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.

Public website mirror

-- RubenDiaz Sep 4 2007

Added:
>
>
 
META TOPICMOVED by="KakoliMitra" date="1234475834" from="Main.CemPPGridsCSA" to="Main.PPCarbonSandwich"

Revision 812 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocGrids"
NYSBC|Cryo-EM P&P: Grid preparation - Carbon Sandwich

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
Changed:
<
<
CEMfac Home NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
>
>
CEMfac Home NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
 

Grid preparation

Protocols - Carbon Sandwich

Contents

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • 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.

Public website mirror

-- RubenDiaz Sep 4 2007

META TOPICMOVED by="KakoliMitra" date="1234475834" from="Main.CemPPGridsCSA" to="Main.PPCarbonSandwich"

Revision 712 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocGrids"
NYSBC|Cryo-EM P&P: Grid preparation - Carbon Sandwich

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
Changed:
<
<
CEMfac Home? NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
>
>
CEMfac Home NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
 

Grid preparation

Protocols - Carbon Sandwich

Contents

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • 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.

Public website mirror

-- RubenDiaz Sep 4 2007

META TOPICMOVED by="KakoliMitra" date="1234475834" from="Main.CemPPGridsCSA" to="Main.PPCarbonSandwich"

Revision 612 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocGrids"
NYSBC|Cryo-EM P&P: Grid preparation - Carbon Sandwich

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website

Grid preparation

Protocols - Carbon Sandwich

Contents

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • 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.

Public website mirror

-- RubenDiaz Sep 4 2007

Changed:
<
<
META TOPICMOVED by="KakoliMitra" date="1234475079" from="Main.CarbonSandwich" to="Main.CemPPGridsCSA"
>
>
META TOPICMOVED by="KakoliMitra" date="1234475834" from="Main.CemPPGridsCSA" to="Main.PPCarbonSandwich"
 

Revision 512 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocGrids"
NYSBC|Cryo-EM P&P: Grid preparation - Carbon Sandwich

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website

Grid preparation

Protocols - Carbon Sandwich

Contents

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • 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.

Public website mirror

-- RubenDiaz Sep 4 2007

Added:
>
>
META TOPICMOVED by="KakoliMitra" date="1234475079" from="Main.CarbonSandwich" to="Main.CemPPGridsCSA"
 

Revision 412 Feb 2009 - Main.KakoliMitra

Changed:
<
<
META TOPICPARENT name="SamplePreparation"
>
>
META TOPICPARENT name="CemProtocGrids"
  NYSBC|Cryo-EM P&P: Grid preparation - Carbon Sandwich

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website

Grid preparation

Protocols - Carbon Sandwich

Contents

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • 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.

Public website mirror

-- RubenDiaz Sep 4 2007

Revision 312 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="SamplePreparation"
Changed:
<
<		CarbonSandwich
>
>
Added:
>
>		 NYSBC|Cryo-EM P&P: Grid preparation - Carbon Sandwich
 
Changed:
<
<
>
>
Added:
>
>		Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
 
Added:
>
>		 Grid preparation

Protocols - Carbon Sandwich

Contents

 

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • 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.
Changed:
<
<
>
>
Added:
>
>		Public website mirror
 
Changed:
<
<		Visit Cryo EM Web site at http://www.nysbc.org/facilities/CEM
>
>
 -- RubenDiaz Sep 4 2007
Deleted:
<
<
 

Revision 207 Dec 2007 - Main.KdDerr

 
META TOPICPARENT name="SamplePreparation"
CarbonSandwich

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • Pipette
  • Filter paper
  • Small weight boat

Procedure:

Changed:
<
<
>
>
  • 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.

Visit Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- RubenDiaz Sep 4 2007

Revision 104 Sep 2007 - Main.RubenDiaz

 
META TOPICPARENT name="SamplePreparation"
CarbonSandwich

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • CarbonCoatedGrid?
  • CarbonCoatedMica?
  • Pipette
  • Filter paper
  • Small weight boat

Procedure:

  • GlowDischarge 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.

Visit Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- RubenDiaz Sep 4 2007

View topic  |  History: r14 < r13 < r12 < r11  |  More topic actions...
 
Copyright © by the contributing authors. All material on this collaboration platform is the property of the contributing authors.
Ideas, requests, problems regarding this intranet, Send feedback