大肠杆菌cDNA文库构建与质量分析.docVIP

大肠杆菌cDNA文库构建与质量分析 　　摘 要 采用Stratagene公司的cDNA文库构建试剂盒构建大肠杆菌的cDNA文库，从中筛选出与RNase Ⅲ有相互作用的蛋白，为研究RNase Ⅲ蛋白在原核生物中的作用奠定基础.用Trizol试剂提取大肠杆菌的总RNA，在E.coli poly（A） Polymerase作用下给mRNA特异性地加上poly（A）尾，分离纯化mRNA，利用mRNA作为模板反转录合成双链DNA.经pfx酶补平双链末端，在两端加上EcoRⅠ接头，XhoⅠ酶切消化产生粘性末端.用CHROMA SPIN400 column分级分离纯化cDNA片段，与pTRG XR载体连接后，电击转入XL1BLUE MRF′菌电转感受态中.得到原始文库，经计算文库的容量达到3×106个克隆.用PCR方法测得文库的重组率为100%，插入片段的平均长度基本位于300 bp以上，测定放大文库的滴度为：3.15×1010pfu/mL.说明构建的大肠杆菌cDNA 文库质量比较高，适合用于筛选目的基因克隆. 　　关键词 大肠杆菌；细菌双杂交；cDNA文库 　　中图分类号 Q78 文献标识码 A 文章编号2016 　　Construction of a cDNA Library of Escherichia Coli and Its Quality Analysis 　　XIAO Ye， WANG Tingting， XIANG Shuanglin* 　　（College of Life Science， Hunan Normal University， Changsha 410081， China） 　　Abstract To investigate the role of RNase Ⅲ in prokaryote， a cDNA library of Escherichia coli was constructed using the cDNA library construction kit purchased from Stratagene， which could be further used for identifying RNase Ⅲ interactingproteins. In this study， the cDNA library of E.coli was constructed using the following method： the total RNA of E.coli was extracted using Trizol kit， and the poly（A） tail was specifically added to mRNA with E.coli poly（A） Polymerase. After that， mRNA was separated， purified， and reversetranscripted into doublestrand DNA. The dscDNA termini was blunted with pfu DNA polymerase. The blunted cDNAs were added to EcoRⅠ adaptors and then digested by XhoⅠ. The cDNA size was fractionated by CHROMA SPIN400 column. These purified fragments were ligased into the pTRG XR vector and transformed into XL1BLUE MRF′ competent cells， which generated the oriental unamplified cDNA library. The unamplified library contained about 3×106 recombinants. The PCR results showed that the percentage of positive recombinant clones was 100%. The length of the inserted cDNA fragment was over 300 bp.The titer of the amplified library was 3.15×1010pfu/mL.The quality of the constructed E.coli library was excellent and helpful to screen new ge