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A protocol for setting up nanolitre sitting-drop vapour-diffusion experiments is reported. The procedure uses standard crystallization screening kits and 96-well crystallization plates. Reservoir solutions are transferred from 96-deep-well blocks to crystallization plates in a single step with a Robbins-Hydra pipettor. Nanolitre droplets of protein as well as reservoir solution are dispensed by a Cartesian pipetting instrument. Experiments have been carried out to characterize the performance of this instrument. Adaptations to the Cartesian, which include an anti-evaporation cover plate, are described and tested. The protocol was designed for a high-throughput facility, but can be used in any standard crystallography laboratory.

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