摘要
目的:从大肠杆菌克隆子中提取和纯化质粒DNApPC41和pcDNA3。方法:采用碱裂解法将质粒DNA从菌体细胞中分离出来,进一步用聚乙二醇沉淀法、透析袋电洗脱法、低溶点胶回收法、玻璃纤维柱层析法纯化质粒DNA。用分光光度法测其A260、A280值,确定其浓度和纯度。通过酶切分析确定其质粒大小。结果:4种方法获得的质粒DNA浓度为0.12～0.24g/L,聚乙二醇沉淀法、透析袋电洗脱法、低溶点胶回收法和玻璃纤维柱层析法的A260/A280比值依次为1.9、2.2、2.2、2.6。酶切鉴定质粒pPC41大小为10.6kb,pcDNA3大小为5.4kb。结论:4种方法均能有效地从大肠杆菌克隆子中提取和纯化得到质粒DNA。其中玻璃纤维柱层析法获得的质粒DNA纯度最高,是获取高纯度质粒DNA的有效方法之一。
Objective: To explore one method getting highly purified plasmid DNA and costing low.Methods: According to principle of extraction and purification plasmid DNA of glass fiber chromatographic column, its operation sequence was improved so that disposable glass fiber chromatographic column could be used repeatedly. The concentration and purity of plasmid DNA obtained by 12 times repeated using glass fiber chromatographic column were determined by spectrophotometry. Moreover, Those plasmid DNA obtained were tested by restriction enzyme and agarose gel electrophoresis, Ligation in vitro and transformation test. Results: 12 times repeated use of glass fiber chromatographic column made no significant difference to the concentration and purity of obtained plasmid DNA(P>0.05), and plasmid DNA was completely digested by restriction enzyme. The digested fragments were well ligated together by the action of T4 DNA Ligase in ATP ligating buffer in vitro and ligated DNA was well transformed into E.coli. Conclusion: Repeated use glass fiber chromatographic column can obtain a lot of highly purified plasmid DNA and cost is low. Digestion, ligation and transformation of those plasmid DNA are not affected. The results may be significant in molecular cloning and study of gene vaccination.\;
出处
《华西口腔医学杂志》
CAS
CSCD
北大核心
1999年第4期361-363,I016,共4页
West China Journal of Stomatology
基金
国家自然科学基金!资助项目 (编号 3 9770 797
关键词
变形链球菌
表面蛋白抗原
质粒
载体构建
龋齿
column chromatography\ \ plasmid DNA\ \ extraction and purification
