【摘要】 目的:分析大肠杆菌JM109感受态形成因素,提高转化效率。方法:采用不同生长状态、不同转化溶液、不同保存时间及热激处理时间的细菌制备感受态,分析转化效率。结果:以20mmol/L MgCl2+80mmol/L CaCl2为处理液,经活化培养OD600为0.82的菌液制备感受态细胞,4℃放置12~24h之内,42℃热激处理60s,转化效率最高,可达9.8×106~1.2×107cfu/μg DNA(pUC19)。随着质粒长度增加,转化效率下降。结论:感受态细胞形成与生长状态关系密切,金属离子、有机溶剂对感受态的形成影响显著。感受态形成过程中,细胞可能发生了一系列的生理变化。更多还原

【Abstract】 Objective:Analyzing the affecting factors of forming competent cells of Escherichia coli JM109 to increase transformation efficiency.Methods: A series of Escherichia coli JM109 competent cells were produced in different conditions,including growth status,transformation solutions,storing time at 4℃,activating time at 42℃,plasmid length.Different effects on transformation ability of competent Escherichia coli JM109 were evaluated.Result: The highest transformation ability was obtained when the activated cells was cultured to an OD(600) 0.82,then prepared with 20mmol/L MgCl2 and 80 mmol/L CaCl2,kept at 4℃ between 12h to 24h,hot shocked at 42℃ for 60s.The transformation efficiency for optimized protocol was found to be as high as between 9.8×106 to 1.2×107cfu/μg DNA(pUC19).The lower efficiency was,the longer the length of the plasmid was.Conclusion: The formation of the competent cells was concerned with growth status,some metal ions and organic solutions.The physiologic change could take place in the process of forming competent cells.更多还原