High-efficiency electroporation by freezing intact yeast cells with addition of calcium.

We developed a novel freezing method to generate competent cells of Schizosaccharomyces pombe and Saccharomyces cerevisiae prior to electroporation. Freezing the intact cells in sorbitol with the addition of calcium at -80 degrees C allowed us to improve the transformation efficiency after freezing and thawing. The optimum concentration of CaCl(2) was found to be 5-10 mM. The addition of other cations had no effect on the efficiency, while the addition of calcium meant that a broad concentration of sorbitol (0.6-2.5 M) could be used, independent of strain. Moreover, increasing the cell concentration to 2 x 10(9) cells/ml during an applied electric pulse further increased the efficiency after freezing and resulted in a wide range of electric field strength (9.0-11.5 kV/cm). Therefore, there was no need to optimize both the concentration of cryoprotectant and the electric field strength of the applied pulse. This procedure for electroporation allows the frozen competent cells to be stored long-term without any significant loss of efficiency.