摘要
目的对含野生型大鼠Rab5a基因载体pcDNA3-Rab5(a)WT进行体外转化、扩增及鉴定,为基于Rab5a的相关研究提供高纯度、高丰度的表达载体。方法取pcDNA3-Rab5(a)WT质粒转化DH5α感受态菌,再接种到含氨苄青霉素的LB琼脂培养板进行抗性筛选,对挑取的阳性克隆进行LB液体培养基培养后抽提质粒,用超微量核酸蛋白测定仪测定提取质粒的纯度和浓度,并取样进行琼脂糖凝胶电泳及PCR鉴定。结果经转化后获得了大量抗氨苄青霉素的阳性克隆菌落,挑取阳性克隆并经培养后提取的pcDNA3-Rab5(a)WT质粒均为浓度高、纯度高、超螺旋结构为主的质粒。pcDNA3-Rab5(a)WT经PCR后得到Rab5a基因。结论成功转化并扩增了pcDNA3-Rab5(a)WT,为Rab5的相关研究提供了高质量的表达载体。
Objective To transform, amplify and identify pcDNA3-RabS（a）WT plasmid carrying Rat wild-type RabJa gene, providing high quality of expression plasmid for Rab5a interrelated studies. Methods pcDNA3-Rab5（a） WT was transformed into DHSa competent cell and cultured in LB agar plate containing ampicillin. Positive clones were selected and cultured in LB liquid medium before plasmid isolating. The purity and concentration of isolated pcDNA3-Rab5 （a） WT plasmid from clone culture were analyzed by NanoDrop 2000/2000c Spectrophotometers. And the plasmid were identified by PCR using Rat RabSa primers. Results The purity and concentration of all 5 isolated pcDNA3-Rab5（a）WT Plasmid samples from clone culture were high and the super helix structure of pcDNA3-RabS（a） WT Plasmid were rich. The plasmid PCR product were 325bp-length RabSa gene. Conclusion Successfully transforming and acquiring high purity and concentration of peDNA3-Rab5 （a）WT plasmid, which provide necessary materials for Rab5 related studies.
出处
《中华肺部疾病杂志（电子版）》
CAS
2013年第6期13-15,共3页
Chinese Journal of Lung Diseases(Electronic Edition)
基金
国家自然科学基金面上项目(81170066)
