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Sample Prep Room Protocol

Location:

We are located at B02A in the basement of Seely G. Mudd, at 250 Longwood Avenue, Boston MA 02115. 

General Sample Prep Room rules:

  1. After you complete an initial vitrobot training, Sign up on PPMS calendars in order to reserve the room, before using the Vitrobots.
  2. All users are expected to have their own grids, grid handling tweezers, grid buttons and long term cryo-grid storage (email cryoEM@hms.harvard.edu for a list of reccommended supplies). Vitrobot tweezers are shared by all users and should be handled with extreme care. It is reccommended to purchase your own set of vitrobot tweezers, but not neccessary. 
  3. 4L LN2 dewars must remain in/return to the sample prep room.
    • LN2 may be obtained from the dewar in the hallway
  4. Clean up after you finish plunging your samples and put all materials back where you found them.
  5. Properly dispose of any BSL-2 waste
  6. Fill out the log book when you are finished and note any problems.
  7. Complete the vitrobot sign-off checklist in the logbook (Turn off the vitrobot properly using the 2-step process, remove the humidifier, close the ethane). 
  8. Turn off the lights and shut the door when you leave the room.

 

Vitrobot Operation

If you are new to cryoEM or you want to refresh your knowledge please see cryoem101.org or https://em-learning.com/

Do not hesitate to ask staff for help if you are unsure of anything. Check Log to make sure there are no problems

 

Turn On the Vitrobot(s)

  1. Attach humidifier (dot on front of humidifier must face towards the front of instrument).
  2. Assure the o-ring is seated properly.
  3. Attach the humidifier to power supply cord.
  4. Fill humidifier with ~40 mL of milliQ water. Remove 10 mL of air.
  5. Turn ON Vitrobot using the switch on the back right side of the instrument.
  6. On UI Screen select the Console tab: set humidifier to 100% and set your desired temperature (~4-22 degrees Celsius).
  7. Go to Options tab to change your blot parameters. Use the logbook to see a record of what other folks are doing.
  8. Open the chamber and mount the plotting paper onto each pad with clipping rings.
  9. Fill the 4L liquid nitrogen hand dewar using the larger tanks in the hallway (make sure to avoid frosty contamination!)
  10. Cool down your vitrification pot with liquid nitrogen ~5-10 minutes before condensing ethane.
  11. Use the Spider while cooling the freezing cup to ensure the ethane cup is cold enough.
    •  Do not leave excess nitrogen in the ethane cup before dispensing ethane and avoid splashing nitrogen into your condensed ethane.
  12. Glow discharge grids: Make sure settings are correct. If you change any, remember to change them back.
  13. Make sure to handle the community vitrobot tweezers with care or, alternatively, purchase your own.
  14. Remove spider from the freezing cup before you prepare and store your grids.

 

Shutting down the Vitrobot(s)

  1. When finished, remove the tweezers and shut down the Vitrobot.
    • This is a 2-step process: Press Exit on the GUI screen, then make sure to turn off the physical switch on the back of the instrument.
  2. Unplug the humidifier power cord and remove the humidifier from the instrument.
    • To remove the power cord, push UP on the gasket button while pulling DOWN on the plug (never force this, ask Staff for help if you cannot remove it).
    • Rotate humidifier clockwise to remove from the instrument.
    • Empty the excess water into the waste water container.
    • Be careful to not lose the O-ring for the humidifier in the waste container.
  3. Humidifier must be left in the designated box, empty for the next user.
  4. Take off blotting paper and place clip rings back inside chamber.
  5. Excess LN2 and liquid ethane should be disposed of in the styrofoam box.
  6. Fill out log­book.
  7. Tightly close both ethane valves and turn off the light before leaving the room.
  8. Put everything back where you got it
  9. If you encounter any problems or think anything is strange with the Vitrobots or sample prep room, please contact any member of the staff before continuing your work (email contact below). CryoEM staff members are generally available on-site Monday-Friday between the hours of 8am-5pm, and on-call in case of emergencies.

 

Loss of privilege warning: If any of the tools or instruments in the room are misplaced, missing, or broken and do not get reported, your access to the sample prep room will be revoked. Under no circumstances should you touch any of the tools in the staff grid clipping area.

 

After Hours Contact: cryoem@crystal.harvard.edu