Instrument access and training
Contact Mariena and/or Darrah for cryoEM related inquiries. Typically we’ll meet to discuss your goals, sample(s), and timeline. Darrah and Mariena have offices in Clark Hall or PSB. Once the goals of the project are established, new users should request an account in FOM for access to instrument calendar(s) and bookings (do not request instrument/booking access until you have discussed your project with a manager). The goal is for users to learn how to prepare (stain and freeze) samples and perform data collection as independent operators.
The typical workflow is as follows:
- Meet with managers to discuss project(s), timeline and goals (in-person or remotely).
- Request access to FOM. While waiting for your FOM account, please read and complete the orientation form for PSB and provide the requested materials to the appropriate facility personnel.
- Once your account is created, coordinate with Mariena to perform negative stain experiment(s) for initial visualization and sample characterization on the BioTwin.
- Following negative stain sample evaluation, the user(s) should optimize the amount of sample (>1mg/mL for cryo), dispersity,and quality before requesting time on the Arctica microscope.
- Training and data collection on the Arctica.
Important sample prep notes:
- For negative stain: continuous carbon coated grids are provided. For cryo users: you will need to purchase the appropriate grids (ideally Quantifoil R1.2/1.3 Cu or Au). These grids take time to arrive and can be backordered, so plan experiments around their availability.
- Users must ensure samples are fresh off a size-exclusion chromatography column prior to their initial session for negative stain and/or cryo sample preparation.
- The sample should be in a buffer it is normally active in and if using glycerol, that it’s <10%.
- Typical concentrations for sample prep by negative stain range from 0.2mg/mL to 1mg/mL.
- For negative stain, avoid the use of phosphate-contaning buffers.
- For single particle cryo, the concentration should be at least 1mg/mL, but this may be vary by sample and buffer and other additives.
- If bringing lipids or vesicle samples, and using a method such as NTA for quantification, ensure the particle concentration is at least in the 1010 range (per mL). Users should expect only a handful of lipid vesicles/particles per image.
New users should arrange to handle and store large volumes of data produced during SPA data collection. CCMR does not provide long-term data storage. Users should also ensure they possess the computational capabilities to process and analyze data. One popular option is requesting membership to SBGrid.