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 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.
    • 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.

 

Using the Vitrobot(s):

  1. A general refresher of how to use the vitrobot can be found at this link: Vitrobot Review; do not hesitate to ask staff for help if you are unsure of anything.
  2. Attach the empty humidifier to the Vitrobot and attach the humidifier's power supply. Assure the o-ring is seated properly. 
  3. Fill the humidifier with 40 mL of Milli-Q water, leave the syringe in place, and then remove 10 mL of air.
  4. Turn on the Vitrobot via the power switch on the back.
  5. When dispensing ethane, be sure to have a 1 mL pipet tip on the end of the trigger. Do not adjust the flow rate.
  6. Excess LN2 and liquid ethane should be disposed of in the styrofoam box.
  7. When finished, remove the tweezers and shut down the Vitrobot.
    • This is a 2-step process: Press EXIT On the GUI screen, then turn off the physical switch on the back. 
  8. Unplug and remove the humidifier; empty the excess water into the waste water container. Be careful to not lose the O-ring for the humidifier in the waste container.
    • Invert the humidifier on a paper towel; use the syringe to remove excess water
  9. Humidifier must be left  in the designated box, empty for the next user.
  10. Tightly close both ethane valves before leaving the room.
  11. 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. CryoEM staff members are generally available on-site Monday-Friday between the hours of 8am-5pm, and on-call in case of emergencies outside of these hours. Emergency contact information will be available on the wall.

 

Using the easiGlow unit:

  1. Be gentle with the easiGlow bell jar - don’t chip the glass. 
  2. The general settings are written on the unit. If you change any of these settings, make sure to change them back when you are finished.
  3. Turn Off the easiGlow unit when you are finished.

 

Loss of privilege warning:

  • Under no circumstances should you touch any of the tools in the grid clipping area of the bench.  If any of the tools are misplaced, missing, or broken, your access to the sample prep room will be revoked.