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
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
|