A rapid and simple procedure for high-efficiency lithium acetate transformation of cryopreserved Schizosaccharomyces pombe cells.
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/microg 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.
Suga
Minoru
M
Department of Materials and Biosystem Engineering, Toyama University, 3190 Gofuku Toyama-City, Toyama 930-8555, Japan. suga@eng.toyama-u.ac.jp
Hatakeyama
Toyomasa
T
D000085:Acetates; D015925:Cryopreservation; D018274:Electroporation / Q000379:methods; D005990:Glycerol; D012440:Saccharomyces / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*; D014170:Transformation, Genetic
D000085:Acetates; C488804:lithium acetate; D005990:Glycerol