Interested in EM?
Using the NYSBC
Principles & Protocols
NYSBC Equipment
Seminars & Courses
Publications associated with CEM at NYSBC
Jobs Available


Microscope Schedule
Logbook
NYSBC Intranet
NYSBC Contacts
Cryo-EM home



NYSBC home
NMR Facility
X-Ray Facility
Protein Production Facility
Electron Crystallography Center


Cryo-EM Site map


Principles & Protocols



Protocols - Using Carbon Thread in Edwards 306

Jump to Principles


You will need:

  • Carbon thread
  • Thread holder
  • Screwdriver
  • Filled LN2 trap

Setup

  • Vent jar by pressing vent
    • If "Vent" is not allowed (red light on key not lit), press "Start" then press Vent
  • When vented (2MB), remove jar by first disconnecting hinge, then lifting and rotating away
  • Secure carbon thread in holder (cut a ~4cm piece, place between screws and screw down)
  • Attach the holder to rod B&D and grounded rod 7cm from grid
  • Insert sample
  • If desired, fold a piece of filter paper and place near sample -- this will let you see the carbon shadow
  • Hold in place with microscope slide or pennies
  • Lower jar and press "Process"

Coating

  • Move stop over sample to prevent C from coating during step 10
  • Pump down to 10-4
  • Turn dial to #0 (L)
  • Switch LT selector to D (30V)
  • Switch on LT
  • Turn the selector up to # 2 to make the thread glow a few seconds and off gas contamination slowly
    • if thread does not glow, his "Reset" button to allow current to flow
  • Remove stop to coat with C
  • Insert LN2 trap into hole on left side of pumping station. Trap is stored in drawer below station.
  • Add liquid nitrogen to the LN2 trap
  • Allow vacuum to recover to 5x10-6 - may need to be restarted
  • Trip the LT (this prevent current from flowing when you increase the current value in next step)
  • Set selector to 6
  • Press reset & look away
  • Thread will glow brightly until it burns out, leaving a thick layer of carbon on sample.
  • Turn off LT

Shutdown

  • Vent bell jar
  • Remove sample -- carbon-coated grids are ready for immediate use
  • Close jar again and pump down
  • When it reaches fine pumping, press "seal"

7cm from grid and 1cm of thread gives about 6 nm carbon coat

This method is very reproducible


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

-- DavidStokes - 18 Mar 2005