【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。更多还原

【Abstract】 In order to optimize the conditions of construction BAC library,the tr ansformation efficiency of E.coli DH10B was studied in this paper.Our data prov e much higher competence of electroporation（reaches 2.19×10～ 10 cfu/μg p UC19 DNA）when harvesting the cells between an OD₅50 of 0.7～0.8.F ive different electric fi eld strength（from 9 kV/cm to 25 kV/cm）and three different sized plasmid vector DNAs including pUC19 DNA,pECBAC1 DNA and pCLD04541 DNA,as well as three bacte rial 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 stre ngth of plasmid DNAs drop dramatically with increasing size of the DNA.Molecule s of 190 kb transform more than 50-fold less well,on a molar basis,than molec u les of 40 kb.And the optimal voltage gradient is strongly dependent on the diff erent sized molecules,for instance,pUC19 reaches the highest transformation ef ficiency 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 appl ied in large-insert genome library construction.更多还原