Document Detail


High-efficiency electroporation by freezing intact yeast cells with addition of calcium.
MedLine Citation:
PMID:  12684838     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Minoru Suga; Toyomasa Hatakeyama
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Publication Detail:
Type:  Journal Article     Date:  2003-04-09
Journal Detail:
Title:  Current genetics     Volume:  43     ISSN:  0172-8083     ISO Abbreviation:  Curr. Genet.     Publication Date:  2003 Jun 
Date Detail:
Created Date:  2003-05-23     Completed Date:  2004-02-19     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8004904     Medline TA:  Curr Genet     Country:  United States    
Other Details:
Languages:  eng     Pagination:  206-11     Citation Subset:  IM    
Affiliation:
Department of Materials and Biosystem Engineering, Toyama University, 3190 Gofuku Toyama-City, 930-8555 Toyama, Japan. suga@eng.toyama-u.ac.jp
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MeSH Terms
Descriptor/Qualifier:
Calcium Chloride / metabolism*
Cryopreservation*
Electroporation / methods*
Sorbitol
Transformation, Genetic
Yeasts / physiology*
Chemical
Reg. No./Substance:
10043-52-4/Calcium Chloride; 50-70-4/Sorbitol

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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