制备高效大肠杆菌电转化感受态细胞和电转化条件的研究

Preparation of Efficient Electrotransformation Competent Cells of Escherichia coli and Condition of Electrotransformation

旨在建立一种高效大肠杆菌电转化感受态细胞的制备方法,研究了摇瓶装液量,菌体生长阶段,转化电场强度,转化后复苏时间以及感受态细胞存放时间等对转化效率的影响。结果表明,装液量为400 mL/2 L,菌体在OD600值为0.452时收集所制备的感受态细胞,在电场强度12.5 kV/cm条件下电击5 ms,转化后复苏时间2 h,转化效率可达到1010CFU/μg DNA。此条件下制备的感受态细胞转化效率高,质量稳定,重复性好。

The aim of this study was to optimize the preparation of E.coli electrotransformation competent cells.In the present work,the effects of different prepare conditions were systematically investigated to improve the transformation efficiency of the competent cells.The optimal conditions were determined as follows： cultivation in 400 ml/2 L LB medium,collection of the cells at OD600 of 0.452,electrotransformation at 12.5 kV/cm for 5 ms,posttransformation culture for 2 h.Under these conditions,the transformation efficiency could reach 1010 CFU/μg DNA.