一种简单的哺乳动物细胞附着体质粒CaCl2转化方法

目的:建立一种简单经济的哺乳动物细胞附着体质粒还原实验方法。方法:构建附着体载体,转染中国卵巢仓鼠(CHO)细胞和小鼠脑神经瘤细胞Neuro-2a,利用改良的赫特裂解法提取附着体质粒,CaCl2法转化附着质粒至宿主菌大肠杆菌DH5α,再次从DH5α中提取质粒,将转染前后质粒用KpnⅠ/BamHⅠ双酶切和BamHⅠ单酶切,并将转染前后的质粒进行DNA测序分析。结果:与最初转染的质粒相比,还原质粒双酶切和单酶切后的条带大小一致;DNA测序分析表明,转染前后质粒中的插入序列相同。结论:建立了可用于质粒还原实验的简单的CaCl2转化方法。

A Simple Method of CaCl2 Transformation for Episomal Plasmid in the Mammalian Cells

Objective: To establish a simple and economic episomal plasmid rescue approach in the mammalian cells.Methods: The constructed episomal vector was transfected into China ovarian hamster(CHO) cells and mice brain Neurotumor cells.Episomal vector was extracted by the modified Hirt Pyrolysis method,and then transformat ed into the E.coli DH5α by CaCl2,followed by extraction plasmid from the E.coli DH5α once again.Before and af ter transfection,the episomal vectors were digested with KpnⅠ/BamHⅠ and BamHⅠ,and the DNA sequences were analyzed.Results: Compared with the initial transfected plasmid,the extracted episomal plasmids were identi cal through detection by restriction enzyme digestion and DNA sequencing.Conclusion: We established a simple methord of CaCl2 transformation for the plasmid rescue experiment successfully.