【摘要】 通过直接在大肠杆菌碱裂解上清中加入十六烷基三甲基溴化铵(CTAB),优化CTAB与质粒DNA量的比例、质粒DNA选择性释放溶液的选择和TritonX-114的使用,建立了简单、易行的大规模质粒DNA纯化工艺。纯化质粒DNA的质量检测结果显示,CTAB纯化的质粒DNA无菌体RNA污染,菌体基因组DNA、内毒素和蛋白含量分别小于100ng/mg、50EU/mg和10μg/mg质粒DNA,OD260/OD280比值介于1.75~1.85之间,超螺旋质粒DNA的比例大于80%,该工艺纯化的质粒DNA能达到或接近FDA规定的人用质粒DNA的各项指标,整个过程不使用动物源性酶和苯酚、氯仿、无水乙醇等有毒或易燃、易爆试剂,成本低廉,工艺环保。更多还原

【Abstract】 Following Escherichia coli lysis with alkali, cetyltrimethylammonium bromide (CTAB) was directly titrated into the supernatant. An easy and feasible technology for plasmid purification was established with the optimized proportion between the quantity of CTAB and plasmid, combined with the specific solution for DNA release and TritonX-114 for endotoxin removal. Quality detection showed that the purified plasmid was free of contamination of host RNA. The host bacterial genomic DNA, endotoxin and bacterial protein were less than 10 μg, 50 EU and 10 μg per mg plasmids, respectively. The ratio of OD260/OD280 was between 1.75?1.85. Eighty percent of the prepared plasmids were presented in the supercoiled form. The plasmid purified with this technology can satisfy all criteria stipulated by FDA. The main advantages of the technology include the avoidance of animal-derived enzymes such as ribonucleases A, Proteinase K and toxic reagents like chloroform and phenol. In addition, the technology has low cost and no pollution.更多还原