Method for preparing Escherichia coli electrotransformation competent cells by rapid desalting

doi:10.14088/j.cnki.issn0439-8114.2020.17.039

Abstract

Abstract: A similar density gradient centrifuge method was adopted to establish an efficient and fast protocol for Escherichia coli electrotransformation. Angle rotor centrifuge was used to achieve the goal of rapid desalination of cells using 20% glycerol （V/V） /1.5% mannitol （m/V） solution as gradient medium. The ratio of bacteria suspension to gradient medium was 1∶5 （V/V） to form a density gradient, which led to effectively centrifugal desalination. After centrifugation the cells were resuspended in a mixture of 20% glycerol （V/V）/1.5% mannitol （m/V）, sequentially electroporated at 15 kV/cm. This procedure was suitable for effective desalination. Electroporation at room temperature did not improve transformation efficiency. In this study, the electransformation efficiency can reach 4.2×10⁹ CFU/μg DNA only by two centrifugation, which greatly simplifies the experimental procedure.

Key words: electrotransformation, competent cells, desalting, Escherichia coli

CLC Number:

Q812

CAI Song, YANG Dong-cheng, TANG Mei, XIONG Yuan-yuan, WANG Jin-hua, WANG Yong-ze. Method for preparing Escherichia coli electrotransformation competent cells by rapid desalting[J]. HUBEI AGRICULTURAL SCIENCES, 2020, 59(17): 170-174.

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