优化感受态细胞制备方法提高转化效率的研究

Study on competent cell preparation for increasing efficiency of transformation

通过研究不同生长状态、处理溶液、保存时间及热激时间对感受态细胞制备的影响,分析了影响大肠杆菌株JM109和DH5α感受态细胞形成因素,优化了制备感受态细胞的方法。结果表明,OD600为0.43的大肠杆菌菌液经处理液(20 mmol/L MgCl2+80 mmol/L CaCl2)处理,-85℃放置12～14 h,42℃热激处理90 s,转化效率最高,可达5.5×105～6×105 cfu/μg DNA(pSilencer2.1-U6-neo),随着质粒放置时间增加,转化效率下降。

The affecting factors of forming Escherichia coli competent ceils were analyzed to increase transformation efficiency in this paper. A series of competent cells of Escherichia coli were produced in different conditions, including the growth status, the different solutions, the different storing time, hot shock time, plasmid length. And different effects on transformation ability of competent Escherichia coli were evaluated. The result was as follows： The highest transformation ability was obtained when the cells was cultured to an OD600 0. 43, then prepared with 20 mmol/L MgCl2 and 80 mmol/L CaCl2 and kept at -20℃ between 12 h to 14h, hot shock time was at 42℃ for 90 s. The optimized protocol of transformation efficiency waw found to be as high as between5.5×10^5- 6 ×10^5 cfu/μg DNA. The lower efficiency was, the longer the storage time of the plasmid was.