Welcome to the CEMRC Protocols.

Here, you will find a number of useful protocols for: (1) preparing samples for negative stain and cryo-TEM imaging, (2) using preparative equipment, (3) using the microscopes for sample screening and data collection, and (4) data processing and management. This page is always under construction because we are adding and revising protocols based on CEMRC user feedback and facility updates.

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Negative Stain Sample Preparation.

Negative stain TEM is a method for rapidly assessing the quality of your sample for future EM-level imaging experiments. The sample, which can be a purified macromolecule or complex, virus, bacteria, or other object, is applied to an EM grid and embedded in a thin layer of a heavy metal salt solution (stain). The stain improves specimen contrast so that gross and finer details of the sample may be observed.

Below we have provided a primer on negative stain TEM and our basic facility protocol that has been used for preparing negative stain EM grids of proteins, other macromolecules, viruses, and bacteria for downstream imaging in the L120C 120 kV TEM.



Cryo-EM Sample Preparation.

Once you have optimized your sample in the ‘wet-lab’ and examined it by negative stain TEM and/or other means, the next step is to prepare your cryo-EM specimens. Depending on the sample, there are a number of approaches to take for sample freezing (vitrification). Traditionally, most aqueous samples of macromolecules, viruses, small non-adherent cells, and other materials can be vitrified successfully by plunge freezing. Briefly, a few microliters of a sample are applied to an EM grid, blotted with filter paper to produce a thin layer of solution across the EM grid, and then plunge frozen into a cryogen. There are several sample vitrification solutions available in the CEMRC and surrounding laboratories at UW-Madison. The two you will most commonly use for samples prepared by plunge freezing are the TFS Vitrobot and Leica EMGP2.

TFS Vitrobot Mark IV. The Vitrobot is a semi-automated vitrification robot for preparing cryo-EM grids via plunge freezing. The Vitrobot program incorporates a number of user-specified settings that allow for flexibility based upon the requirements of the sample and the user’s needs. Below, we have provided the facility protocol manual for using the Vitrobot and the trainee and trainer checklists we use for signing each user off for independent operation of the Vitrobot. Additional links to information about the Vitrobot and its use are presented in the facility protocol manual.




Leica EMGP2 (coming soon!).

Microscopes and Microscope Operations.

Data Processing.

To access your imaging data using Globus, please refer to this guide.