Difference: PPClaudioHoleyFilms (1 vs. 15)

Revision 1521 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 - Formvar Film

>
>

Protocols - Formvar Holey Film

 

Jump to Principles

This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.
Public website mirror

-- KdDerr - 05 Oct 2006

META TOPICMOVED by="KakoliMitra" date="1234475797" from="Main.CemPPGridsCHF" to="Main.PPClaudioHoleyFilms"

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 - Formvar Film

 

Jump to Principles

This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.
Public website mirror

-- KdDerr - 05 Oct 2006

META TOPICMOVED by="KakoliMitra" date="1234475797" from="Main.CemPPGridsCHF" to="Main.PPClaudioHoleyFilms"

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

Changed:
<
<

Grid preparation

>
>

Protocols - Grid preparation

Jump to Principles

 This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.
Public website mirror

-- KdDerr - 05 Oct 2006

META TOPICMOVED by="KakoliMitra" date="1234475797" from="Main.CemPPGridsCHF" to="Main.PPClaudioHoleyFilms"

Revision 1219 Aug 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtocGrids"
Deleted:
<
<
 

Principles & Protocols

Grid preparation

This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.
Deleted:
<
<

Ready to apply sample

 
Public website mirror

-- KdDerr - 05 Oct 2006

META TOPICMOVED by="KakoliMitra" date="1234475797" from="Main.CemPPGridsCHF" to="Main.PPClaudioHoleyFilms"

Revision 1119 Aug 2009 - Main.KdDerr

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

Principles & Protocols

 
Changed:
<
<
>
>

Grid preparation

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

Grid preparation

Protocols - Claudio Holey Films

Contents

 

This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.

Ready to apply sample

Public website mirror

-- KdDerr - 05 Oct 2006

META TOPICMOVED by="KakoliMitra" date="1234475797" from="Main.CemPPGridsCHF" to="Main.PPClaudioHoleyFilms"

Revision 1012 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocGrids"
NYSBC|Cryo-EM P&P: Grid preparation - Claudio Holey Films

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 - Claudio Holey Films

Contents

This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.

Ready to apply sample

Public website mirror

-- KdDerr - 05 Oct 2006

Added:
>
>
 
META TOPICMOVED by="KakoliMitra" date="1234475797" from="Main.CemPPGridsCHF" to="Main.PPClaudioHoleyFilms"

Revision 912 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocGrids"
NYSBC|Cryo-EM P&P: Grid preparation - Claudio Holey Films

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 - Claudio Holey Films

Contents

This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.

Ready to apply sample

Public website mirror

-- KdDerr - 05 Oct 2006

META TOPICMOVED by="KakoliMitra" date="1234475797" from="Main.CemPPGridsCHF" to="Main.PPClaudioHoleyFilms"

Revision 812 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocGrids"
NYSBC|Cryo-EM P&P: Grid preparation - Claudio Holey Films

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 - Claudio Holey Films

Contents

This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.

Ready to apply sample

Public website mirror

-- KdDerr - 05 Oct 2006

META TOPICMOVED by="KakoliMitra" date="1234475797" from="Main.CemPPGridsCHF" to="Main.PPClaudioHoleyFilms"

Revision 712 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocGrids"
NYSBC|Cryo-EM P&P: Grid preparation - Claudio Holey Films

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 - Claudio Holey Films

Contents

This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.

Ready to apply sample

Public website mirror

-- KdDerr - 05 Oct 2006

Changed:
<
<
META TOPICMOVED by="KakoliMitra" date="1234474957" from="Main.GlycerolHoleyFilms" to="Main.CemPPGridsCHF"
>
>
META TOPICMOVED by="KakoliMitra" date="1234475797" from="Main.CemPPGridsCHF" to="Main.PPClaudioHoleyFilms"
 

Revision 612 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocGrids"
NYSBC|Cryo-EM P&P: Grid preparation - Claudio Holey Films

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 - Claudio Holey Films

Contents

This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.

Ready to apply sample

Public website mirror

-- KdDerr - 05 Oct 2006

Added:
>
>
META TOPICMOVED by="KakoliMitra" date="1234474957" from="Main.GlycerolHoleyFilms" to="Main.CemPPGridsCHF"
 

Revision 512 Feb 2009 - Main.KakoliMitra

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

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 - Claudio Holey Films

Contents

This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.

Ready to apply sample

Public website mirror

-- KdDerr - 05 Oct 2006

Revision 412 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="SamplePreparation"
Changed:
<
<		Contents
>
>
Added:
>
>		 NYSBC|Cryo-EM P&P: Grid preparation - Claudio Holey Films
 
Changed:
<
<

Claudio Holey Grids

>
>
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 - Claudio Holey Films

Contents

 This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.

Necessary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • conical beaker
  • probe sonicator
  • dipping jar
  • crystallizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissolve overnight

Making holey film

  • Place 50 mL of formvar solution in a conical beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to release film
  • float the film on water in a crystallizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissolve holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.

Ready to apply sample

Changed:
<
<
  • Set ALLOWTOPICVIEW =
>
>
Added:
>
>		Public website mirror

 -- KdDerr - 05 Oct 2006

Revision 313 May 2008 - Main.KdDerr

 
META TOPICPARENT name="SamplePreparation"
Contents

Claudio Holey Grids

Changed:
<
<		This method has a sharp edge at the hole as opposed to the beveled thicker edge of triofiol holey films.
>
>		This method has a sharp edge at the hole as opposed to the beveled thicker edge of triafol holey films.
 
Changed:
<
<

Necessiary Supplies

>
>

Necessary Supplies

 
  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
Changed:
<
<
  • concical beaker
>
>
  • conical beaker
 
  • probe sonicator
  • dipping jar
Changed:
<
<
  • crystalizing dish
>
>
  • crystallizing dish
 

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
Changed:
<
<
    • Allow to dissove overnight
>
>
    • Allow to dissolve overnight
 

Making holey film

Changed:
<
<
  • Place 50 mL of formvar solution in a concial beaker
>
>
  • Place 50 mL of formvar solution in a conical beaker
 
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

Changed:
<
<
  • Score grid to relase film
  • float the film on water in a crystalizing dish.
>
>
  • Score grid to release film
  • float the film on water in a crystallizing dish.
 
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
Changed:
<
<
  • Dissovle holes with methanol for 10mins.
>
>
  • Dissolve holes with methanol for 10mins.
 
    • Make sure to avoid covering top of grids with methanol
Changed:
<
<
    • A petri dish with methanol wetted filer paper works well.
  • Allow to dry thouroughly.
>
>
    • A petri dish with methanol whetted filer paper works well.
  • Allow to dry thoroughly.
 
  • Evaporate with carbon.
Changed:
<
<
  • Dissolve plastic away with choloform for 10 minutes
    • Use a petri dish with wetted filter paper agian
  • Evaportate another layer of carbon.

Ready to appy sample

>
>
  • Dissolve plastic away with chloroform for 10 minutes
    • Use a petri dish with whetted filter paper again
  • Evaporate another layer of carbon.

Ready to apply sample

 
  • Set ALLOWTOPICVIEW =

-- KdDerr - 05 Oct 2006

Revision 205 Oct 2006 - Main.KdDerr

 
META TOPICPARENT name="SamplePreparation"
Contents

Claudio Holey Grids

Changed:
<
<		This method has a sharp edge as opposed to the beveled thicker edge of triofiol holey films.
>
>		This method has a sharp edge at the hole as opposed to the beveled thicker edge of triofiol holey films.
 

Necessiary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • concical beaker
  • probe sonicator
  • dipping jar
  • crystalizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissove overnight

Making holey film

  • Place 50 mL of formvar solution in a concial beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to relase film
  • float the film on water in a crystalizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissovle holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol wetted filer paper works well.
  • Allow to dry thouroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with choloform for 10 minutes
    • Use a petri dish with wetted filter paper agian
  • Evaportate another layer of carbon.

Ready to appy sample

  • Set ALLOWTOPICVIEW =

-- KdDerr - 05 Oct 2006

Revision 105 Oct 2006 - Main.KdDerr

 
META TOPICPARENT name="SamplePreparation"
Contents

Claudio Holey Grids

This method has a sharp edge as opposed to the beveled thicker edge of triofiol holey films.

Necessiary Supplies

  • 0.5% Formvar in Chloroform solution.
  • 50% glycerol in water solution.
  • Methanol
  • Chloroform
  • grids
  • Newspaper
  • carbon coater
  • concical beaker
  • probe sonicator
  • dipping jar
  • crystalizing dish

Technique

  • Make 0.5% formvar solution
    • Dissolve 1 g formvar
    • into 200 mL Chloroform
    • Allow to dissove overnight

Making holey film

  • Place 50 mL of formvar solution in a concial beaker
  • add 20 drops of 50% glycerol solution
    • more drops equal larger holes
  • swill contents of conical beaker
  • add contents to dipping jar
  • Probe sonicate dipping jar contents on maximum power for 1 min
    • contents should be warm and milky in color
    • contents should be used to make grid within 1/2 hour of sonication
  • Dip a clean microscope slide into the solution and set to dry staning up on newspaper.
    • microsope slides will keep for years

Applying film to grids

  • Score grid to relase film
  • float the film on water in a crystalizing dish.
  • Place grids on film
  • Pick up film and grids with newspaper.
  • Allow the paper to dry.
  • Dissovle holes with methanol for 10mins.
    • Make sure to avoid covering top of grids with methanol
    • A petri dish with methanol wetted filer paper works well.
  • Allow to dry thouroughly.
  • Evaporate with carbon.
  • Dissolve plastic away with choloform for 10 minutes
    • Use a petri dish with wetted filter paper agian
  • Evaportate another layer of carbon.

Ready to appy sample

  • Set ALLOWTOPICVIEW =

-- KdDerr - 05 Oct 2006

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