汕头大学医学院学报

目的:建立一种高效、快速的大肠杆菌感受态细胞制备及质粒转化的方法。方法:质粒DNA加入感受态细胞,经短暂冰浴和热休克后,直接涂布预热至37℃的平板。以大肠杆菌DH5α为受体菌,研究CaCl2溶液浓度、感受态细胞的存放时间、转化时间及转化后的培养时间等对质粒转化效率的影响。结果:以100 mmol/L CaCl2溶液制备感受态细胞,冰浴转化5 min,经42℃热休克作用90 s后直接涂平板筛选转化子,获得与常规转化方法相当的转化效率。CaCl2溶液浓度和感受态细胞的存放时间对转化效率有明显的影响,而转化时间和转化后的振荡培养时间对转化效率的影响不明显。结论:成功地建立了一种高效、快速的感受态细胞制备及质粒转化的方法。

Objective:To establish an efficient and rapid method for the preparation and transformation of competent cells from Escherichia coli.Methods: The various time incubation of competent cells with plasmids on ice and spreading onto agar plates warmed beforehand to 37 ℃.The different effects of the transformative condition which included CaCl₂ concentration,of the competent cells status,of the various transformative time and of the incubative time on the transformative plasmid were investigated.The host cell was Escherichia coli DH5α.Results: High transformation efficiency was achieved by preparing the competent cells with 100 mmol/L CaCl₂,transforming for 5 minutes,heat shocking after transformation in 42 ℃ for 90 seconds,and then following the direct selection on Amp~+LB plate.The effects of different concentration of CaCl₂ and the competent cells status on the transformative frequency were obvious,but the effects of the transformative time and incubative time after transformation were not significant.Conclusion: An efficient and rapid method for the preparation and transformation of competent cells is successfully established.