A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed. Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells. Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment. A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30uC for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure. It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency. The optimal cell concentration and amount of DNA during the electric pulse were 1r10 9 cells/ml and 10 ng, respectively. The maximum transformation efficiency, 1.2r10 7 transformants/mg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms. Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non-permeating cryoprotectant such as sorbitol.
A rapid, simple, convenient, and highly efficient transformation of the fission yeast Schizosaccharomyces pombe has been developed. Freezing fission yeast cells in glycerol, a permeating cryoprotectant, with lithium acetate improved remarkably the transformation efficiency by one to two orders of magnitude. The optimum concentration of glycerol was found to be 30%, which is higher than that (10-15%) in the conventional cryopreservation of yeast cells. Glycerol not only played a role in cryopreserving the competent cells but also improved the transformation efficiency of the process. The thawed cell suspension with glycerol and lithium acetate was immediately mixed with carrier DNA, plasmid DNA and polyethylene glycol. Next, the mixture was heat shocked and directly spread on a selection plate. This simple procedure yielded more than 10 6 transformants/µg plasmid DNA, reducing the time required to only 20 min in total, including the thawing time. Furthermore, the frozen competent cells were stored long-term for more than 3 months without any significant loss of efficiency.
We have developed a simple method for cryopreserving Schizosaccharomyces pombe and Saccharomyces cerevisiae competent intact cells that permits high transformation efficiency and long‐term storage for electroporation. Transformation efficiency is significantly decreased if intact cells are frozen in common permeating cryoprotectants such as glycerol or dimethyl sulphoxide. On the other hand, we found that a high transformation efficiency could be maintained if the cells were frozen in a non‐permeating cryoprotectant such as sorbitol. The optimum concentration of sorbitol was found in a hypertonic solution of around 2 M. It was also very important to use S. pombe cells grown in minimal medium and S. cerevisiae cells grown in nutrient medium in the exponential growth phase. A slow freezing rate of 10°C/min and a rapid thawing rate of 200°C/min resulted in the highest transformation efficiency. We also found it necessary to wash the thawed cells with 1.0 M of non‐electrolyte sorbitol, since the intracellular electrolytes had leaked as a result of cryoinjury. The frozen competent cells stored at −80°C could be used for more than 9 months without any loss of transformation efficiency. This cryopreservation method for electroporation is simple and useful for routine transformations of intact cells. Frozen competent cells offer the advantages of long‐term storage with high efficiency and freedom from the preparation of fresh competent cells for each transformation. Copyright © 2000 John Wiley & Sons, Ltd.
A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed. Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells. Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment. A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30uC for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure. It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency. The optimal cell concentration and amount of DNA during the electric pulse were 1r10 9 cells/ml and 10 ng, respectively. The maximum transformation efficiency, 1.2r10 7 transformants/mg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms. Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non-permeating cryoprotectant such as sorbitol.
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