Document Detail


Cryopreservation of competent intact yeast cells for efficient electroporation.
MedLine Citation:
PMID:  10870100     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 degrees C/min and a rapid thawing rate of 200 degrees 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 degrees 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.
Authors:
M Suga; M Isobe; T Hatakeyama
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Yeast (Chichester, England)     Volume:  16     ISSN:  0749-503X     ISO Abbreviation:  Yeast     Publication Date:  2000 Jul 
Date Detail:
Created Date:  2000-10-05     Completed Date:  2000-10-05     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8607637     Medline TA:  Yeast     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  889-96     Citation Subset:  IM    
Copyright Information:
Copyright 2000 John Wiley & Sons, Ltd.
Affiliation:
Department of Materials and Biosystem Engineering, Toyama University, 3190 Gofuku Toyama-City, Toyama 930-8555, Japan. suga@eng.toyama-u.ac.jp
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MeSH Terms
Descriptor/Qualifier:
Adenosine Triphosphate / metabolism
Cryopreservation*
Culture Media
Electroporation / methods*
Saccharomyces cerevisiae / genetics*,  growth & development
Schizosaccharomyces / genetics*,  growth & development
Sorbitol / pharmacology
Transformation, Genetic*
Chemical
Reg. No./Substance:
0/Culture Media; 50-70-4/Sorbitol; 56-65-5/Adenosine Triphosphate

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