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Initial crystallization conditions are typically discovered using commercially available sparse-matrix screens. Positive results are then refined using some type of expansion tray. For example, coarse and shallow gradients can be prepared which vary single chemical parameters around the initial conditions in a grid-screen format. Expansion trays are customarily formulated using numerous volume calculations and pipetting stock solutions into individual wells. This tedious process is plagued by pipetting errors, including differences in viscosity, small volumes with large dilutions, evaporation and poor mixing. Here we present a simple method to standardize expansion-tray formats. Instead of using independent well-volume calculations, the initial and final conditions are formulated and gradients (A/B gradients) are prepared using standardized pipetting maps. These step gradients can be prepared by adding decreasing amounts of the initial (A) solution to consecutive wells followed by the addition of the final (B) solution with increasing volume. This simple idea can be applied to both coarse and shallow grids where the pipetting errors are confined within the boundaries defined by the A and B solutions. Programmable electronic pipettes and robotic liquid handlers can be used to prepare the standardized A/B gradients rapidly, regardless of the components, eliminating reprogramming between trays.

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