You have been assigned a Grid Preparation and Screening (GPS) session at NCCAT. What are the next steps?
The workflow for this access category is as follows:
1. If sending samples:
The amount of sample required for 3-4 days of screening and optimization largely depends on the sample itself, the types of challenges it faces, the optimization steps required. As a ballpark starting point, we recommend:
Sample concentration: > 2 mg/ml
Sample volume: > 1 ml
Buffer volume: 100 ml
This is a general recommendation aimed to allow for trying as many screening conditions as possible in 3-4 days, and not a rule. We will work with you and your sample to determine a suitable amount of samples to prepare for the GPS session.
2. If sending grids:
You may send up to 36 grids in 9 buttons/boxes. By default, we will start screening box1 grid1, then box1 grid2, box1 grid3 and box1 grid4. If more than one box is loaded, then we will go to box2 grid1 and so forth.
Over a 2-3-day session, we will aim to screen 20-30 grids as time allows.
1. If you sent samples:
A customized three- or four-day plan for screening and optimizing your samples will be developed together with you during your pre-session meeting.
A staff member will work closely with you when you’re on site to screen and optimize your sample for cryo-EM. In general, we will be aiming for the following workflow:
1. Prepare sample with generic grid-preparation conditions.
2. Image sample on a TEM and analyze images to understand potential issues.
3. Optimize sample preparation conditions.
4. Repeat steps 2-3.
You will have access to all the sample optimization tools available on-site, including carbon and gold grids, graphene grids, Vitrobot, the Chameleon, and Refeyn. We also have a buffer optimization screening kit, and a fully equipped wet lab.
In a three-day visit, we will aim to complete three cycles of the screening workflow with the goal of preparing Krios-ready grids. At the end of your visit, we will review the findings together and put together a plan for next steps.
2. If you sent grids:
Your grids will be screened on a screening microscope. Your data can be viewed by using the Leginon and Appion webtools as detailed below. You can choose to review your data with us after the session.