Training for cryo-specimen preparation with the Vitrobot requires two hands-on sessions preferably within a two-month period. We will train you how to plunge with your sample, so you will be moving forward with your research right away.
Step 1: Introduction
- Before the first hands-on session, watch this video series, particularly the sections that describe specimen plunging (Steps 21-33 can be skipped):
https://www.youtube.com/playlist?list=PL8_xPU5epJdfd5fM2CjQItR-iRlIEIJk8 - After this introduction, you will know what to expect during your first plunging session at the Vitrobot.
Step 2: Observe and Practice
- In the first hands-on session, CryoEM staff will walk you through the steps of Vitrobot setup, shutdown, and use of the plunger. After the trainer demonstrates plunging with a few samples, you will prepare at least another 4 grids.
- After this session, you will know how to safely operate the Vitrobot.
Step 3: Demonstrate
- In the second hands-on session, you will set up the Vitrobot and perform all of the plunging while the trainer accompanies to ensure safe and successful operation, startup, and shutdown.
- After demonstrating that you understand our procedures to operate the Vitrobot, you will be allowed to book and use the instrument independently.
Prepare for your sessions at the Vitrobot:
- Provide your own materials for training sessions #1 and #2, including sample, grids, grid storage buttons, storage tube, tweezers for handling grids, and have access to an LN2 dewar for transport and long-term your specimens. Commonly needed items are listed here:
https://docs.google.com/document/d/1TGmhVGfLqgTTzXOv8nLgYHC9i4M_7urjxYivMQcNCK8/edit?usp=drive_link - Plan experimental conditions for plunging eight (8) grids for each training session using this sheet:
https://www.dropbox.com/s/rcv19vfoumugegq/GridButtonSheet.pdf?dl=0 - Your plunging session will go more smoothly if we discuss your experimental plan ahead of time. Here are a few suggestions:
- Sparse matrix your variables (sample concentration, blot time, blot force, grid type). Once you know what works, you can optimize conditions in subsequent plunging sessions.
- Sample concentration is the most important variable. For globular single particles, the optimal concentration generally falls between 0.3 and 3.0 mg/mL, typically around 1 mg/mL.
- After sample concentration, the next most important variable is foil hole material and hole diameter. 2.0µm holes tend to have thinner ice in the center than 1.2 µm holes.
Reach out to cryoEM_staff@mit.edu if you have any questions!