PCR法快速筛选重组克隆方法的改进  

A MODIFICATION OF RAPID PCR SCREENING FOR RECOMBINANT CLONE

作　　者：毛伟征[1] 孙道升[2] 司春祥[2] 隋爱华[3] 刘湘萍[3] 陈栋[1] 吕振华[3] 

机构地区：[1]青岛大学医学院附属医院普外科,山东青岛266003 [2]青岛海慈医院检验科 [3]青岛大学医学院附属医院分子生物学中心实验室

出　　处：《青岛大学医学院学报》2003年第3期228-230,共3页Acta Academiae Medicinae Qingdao Universitatis

摘　　要：①目的　以PCR法在菌液中快速筛选重组克隆。②方法　提取小鼠巨噬细胞总RNA ,反转录PCR(RT PCR)获得肿瘤坏死因子 α(TNF α)基因片段 ,TA克隆连接质粒载体、转化大肠杆菌JM10 9。在大肠杆菌培养中的不同时间取样测定大肠杆菌A60 0 值及计数。以不同数量的大肠杆菌为模板进行系列PCR扩增 ,获得适宜PCR扩增条件的大肠杆菌数。③结果　以菌液为模板进行PCR能够快速筛选重组体 ,大肠杆菌模板数在 12 5～ 2 5 0个范围内 ,PCR对阳性克隆的扩增可获得良好结果 ;获得常规培养条件下转化大肠杆菌的生长曲线 ,A60 0 值与大肠杆菌计数间存在显著正相关 (r=0 .93,P <0 .0 0 1)。④结论　通过测定大肠杆菌培养液A60 0 值推算大肠杆菌数量 ,以适宜的大肠杆菌模板行PCR法筛选重组克隆 。Objective\ To establish a rapid screening of recombinant clones by PCR.\ Methods\ Total RNA of mouse microphages was extracted. TNF genes were acquired by RT PCR. JM109, E.coli transformed by recombinant plasmid was inoculated. At a series of time spots, diluted 1 L of medium was plated onto LB agar plate. Meanwhile, A 600 of media was detected with spectrophotometer. Colonies on agar were counted. Media with different amount of E.coli were used as a template, a series of PCR carried out. \ Results\ PCR screening showed that an optimized result could be obtained with templates of media with E. coli amount ranged from 125-250. The growth curve of transformed E.coli was set up. There was a significant linear relationship between colonies and A 600 from 4.5 to 7.0 hours after inoculation.\ Conclusion\ The amount of E.coli can be calculated through detecting A 600 of the media. It is a fast and efficient way to screen recombinant clones with PCR ,which takes a proper amount of E. coli as its template.

关 键 词：PCR法 重组体 筛选 聚合酶链反应 克隆方法 大肠杆菌 

分 类 号：Q503[生物学—生物化学]