Biochemisches Institut

Experimental Setup

Scheduling the crystallization experiments:
The CORE user account allows the researcher to schedule the crystallization experiments by accessing the crystallization calendar and reserve date, time and the number of plates, including the experimental setup temperature. This will make sure PCC is ready for your sample and the protein will be processed at the scheduled time without any delay. When booking the crystallization experiments please keep in mind that PCC's capacity  is limited to a maximum of 16 plates per half day.

Setting up the crystallization experiments
The CORE user account allows the researcher accessing the RockMaker application through the Citrix Workspace and setting up the crystallization experiments prior delivering the protein samples. During the first visit at PCC a personal login will be created for accessing the RockMaker application. At the same time, the researcher is given instructions on how to efficiently set up and organize the crystallization experiments using the RockMaker application, specifying the following information.

Screen and Reservoir solution:
All initial crystallization screens offered by PCC are stored in deep well master blocks at 4°C and are available to the researcher for their experiments.

Crystallization plate:
The standard plates used for all vapor diffusion crystallization experiments are either the SWISSCI UVXPO 2 Lens crystallization plate (Cat# UVXPO-2LENS) or the ARI Intelli-Plate 96-3 LVR (Cat# 102-0001-03) (Material/Equipment)..

Setting up the nano-drop experiments:
The Aquarius liquid handler transfers 75ul of the 96 screening solutions from a deep well master block into the reservoir wells of the SWISSCI UVXPO- or the ARI Intelli-plate.

The standard setup temperature of the crystallization experiment is room temperature (~21°C). For temperature labile protein samples the researcher can request 4°C as the setup temperature of the crystallization experiment. The 4°C setup temperature needs to be stated when scheduling the experiment in the  crystallization calendar.
Depending the setup temperature, one of the two nano-drop liquid handlers – Phoenix (20°C) or Gryphon-LCP (4°C) – pipettes the vapor diffusion crystallization experiment. The researcher specifies the drop volumes for each of the two or three crystallization wells generally ranging between 100nl and 250nl for the protein sample and for the reservoir solution. This option allows the researcher to explore the protein/mother liquor ratio as an additional screening variable.

Alternatively up to two or three different protein samples can be dispensed on one crystallization plate.
The protein sample volume required for setting up crystallization plates can be found in Sample Volume.
The Crystal Clear sealing tape from Hampton Research (Cat# HR4-506) is used to seal all sitting drop crystallization experiments prior incubation.

For custom- and refinement screens the researcher has to provide all stock-solutions needed for the preparation to PCC three days ahead of the scheduled crystallization experiment. Generally a volume of 30-50ml (preferably in a 50ml Falcon® tube) is sufficient for all custom screens. “Read-to-go” crystallization screens provided by the researcher have to be in the 96-well standard format.

The following review provides a summary of the current methods and technologies applied in automated platforms for the setup of initial and follow-up crystallization experiments:
P. Shaw Stewart and J. Mueller-Dieckmann
Automation in biological crystallization.
Acta Cryst. (2014). F70

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