DH10B菌株高效电转化条件探究  

The Study of Optimal Conditions of Electroporation in Escherichia coli DH10B Strain

作　　者：张洋[1] 王志强[2] 刘斌[1] 张晓军[1] 姜枫[3] 相建海[1] 

机构地区：[1]中国科学院海洋研究所 [2]北京华大基因组研究中心 [3]北京华大基因组研究中心,北京101300

出　　处：《生物工程学报》2007年第2期347-351,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金资助(No.30230280;30200213);国家高技术研究与发展项目基金资助(No.2005AA626014)~~

摘　　要：以pUC19、pECBAC1、pCLD04541DNA以及3个不同大小的BACDNA为材料,研究了E.coli DH10B菌株在5个不同脉冲电场下的转化效率。研究发现,随着DNA片段大小的增加,最高转化效率和最适场强迅速减小。利用DH10B细胞转化pUC19 DNA的最适场强是21kV/cm,而190kb BAC DNA仅为13kV/cm;在最适场强下,40kb BAC DNA的转化效率约是190kb BAC DNA的50倍。通过大量数据绘制了不同因素影响下转化效率的变化曲线,优化了E.coli DH10B菌株电转化条件,为质粒的重组转化以及大片段基因组文库的构建奠定了基础。In order to optimize the conditions of construction BAC library, the transformation efficiency of E. coli DH10B was studied in this paper. Our data prove much higher competence of electroperation （reaches 2.19 × 10^10 cfu/μg pUC19 DNA） when harvesting the cells between an OD550 of 0.7 - 0.8. Five different electric field strength （from 9 kV/cm to 25 kV/cm） and three different sized plasmid vector DNAs including pUC19 DNA, pECBAC1 DNA and pCL1）（M541 DNA, as well as three bacterial artificial chromosomes （BACs） ranging from 40 to 190 kb and their mixture were used to discover the transformation efficiency changes under various conditions. Our data show maximum transformation efficiency and optimal electric field strength of plasmid DNAs drop dramatically with increasing size of the DNA. Molecules of 190 kb transform more than 50-fold less well, on a molar basis, than molecules of 40 kb. And the optimal voltage gradient is strongly dependent on the different sized molecules, for instance, pUC19 reaches the highest transformation efficiency at 21 kV/cm, while the 180 kb BAC DNA gets its best efficiency at 13 kV/cm. This paper demonstrates that conditions may be selected which increase the average size of BAC clones generated by electroporation and could be widely applied in large-insert genome library construction.

关 键 词：感受态细胞 电转化 转化效率 脉冲电场强度 质粒DNA 

分 类 号：Q78[生物学—分子生物学]