大肠杆菌DH5a菌株高效电击转化条件的优化

Optimization of the High-efficiency Electroporation Transformation Conditionsfor Escherichia coli DH5a Strain

为探索大肠杆菌DH5a高效电击转化条件,通过对电击转化条件中细胞生长状态、DNA加入量、感受态细胞体积、电场强度、速冻和恢复时间等关键因素进行优化,筛选出大肠杆菌DH5a高效电击转化条件。结果表明:大肠杆菌在细胞生长状态OD_(600)值为0.6时制备感受态细胞,按每管50μL体积分装、-80℃乙醇速冻后,加入10 pg pUC 19质粒DNA,在20 kv·cm^(-1)电场强度、电容25μF、电阻200Ω条件下电击转化,转化后恢复培养60 min,转化效率达10~(10) cfu·μg^(-1) DNA以上水平,可满足基因文库构建、抗体库筛选等试验需求。

In order to explore the conditions of high-efficiency electroporation transformation for Escherichia coli DH5a,the key factors in the conditions of electroporation transformation such as the cell growth state,the adding quantity of DNA,the volume of competent cells,electric field intensity,quick freezing and recovery time were optimized,thus to screen the conditions of high-efficiency electroporation transformation for Escherichia coli DH5a.The results showed that:the competent cells of Escherichia coli were prepared when the OD 600 value of the cell growth state was 0.6.The competent cells were packed in 50μL per tube,and frozen in ethanol at-80℃.Then,10 pg pUC 19 plasmid DNA was added,and the cells were transformed by electric shock under the conditions of 20 kv·cm^(-1)electric field intensity,25μF capacitance and 200Ωresistance.After the transformation,the cells were cultured for 60 min.The transformation efficiency was above 1010 cfu·μg^(-1)DNA,which could meet the needs of genomic library construction and antibody library screening.