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Cell transfer by filtration: evaluation of protocols for transformational competence
P Williams1, W L Hung, R J Redfield
1Department of Zoology, University of British Columbia, Vancouver, Canada.
FEMS Microbiology Letters
|April 1, 1996
Summary
Filtration using membrane filters replaces time-consuming centrifugation in microbiological protocols. This method yields faster, highly competent cells for Escherichia coli, Haemophilus influenzae, and Saccharomyces cerevisiae, maintaining sterility.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Cell collection and washing via centrifugation is a standard but inefficient step in many microbiological protocols.
- Maintaining sterility during cell preparation is crucial for accurate experimental results.
- Existing centrifugation methods are time-consuming and can be inconvenient, particularly in sterile environments.
Purpose of the Study:
- To evaluate the efficacy of membrane filtration as a replacement for centrifugation in cell preparation.
- To determine if filtration improves the speed and efficiency of obtaining competent cells.
- To assess the impact of filtration on transformation efficiency in key microbial models.
Main Methods:
- Standard transformation competence protocols for Escherichia coli, Haemophilus influenzae, and Saccharomyces cerevisiae were modified.
- Centrifugation steps for cell collection and washing were replaced with filtration onto disposable membrane filters.
- Transformation efficiencies of filtered cells were compared to those prepared by traditional centrifugation.
Main Results:
- Filtration efficiently replaced centrifugation in the tested protocols.
- Filtered cells achieved competence sooner compared to cells prepared by centrifugation.
- Transformation efficiencies for filtered cells were comparable or higher than those obtained via centrifugation.
- The filtration method facilitated thorough washing and rapid, gentle cell resuspension without compromising sterility.
Conclusions:
- Membrane filtration is a superior alternative to centrifugation for preparing microbial cells for transformation.
- This filtration technique offers significant advantages in terms of speed, efficiency, and cell viability.
- The method is adaptable for various applications, including metabolic studies and electroporation preparation, while maintaining sterile conditions.