人胃癌混合组织cDNA文库的快速构建和鉴定

Rapid construction and identification of a cDNA library from human gastric cancer compound tissues

摘要: 目的: 构建人胃癌混合组织cDNA文库,筛选克隆胃癌相关基因。方法: 以纯化的poly (A)+RNA为模板,含NotⅠ切点的Oligo (dT)15为引物,利用改良的cDNA快速合成方法,反转录合成cDNA第一、二链,连接连接子,经层析柱纯化去除小片段和多余连接子后,重组于噬菌体载体,行体外包装后以大肠杆菌Y10 90行滴度测试及扩增后,建成cDNA文库。结果: 构建的cDNA文库,含3 2× 10 ⁶重组子,重组率为96%。扩增后文库的滴度达1 2× 10 ¹² pfu/L。结论: 成功地构建了人胃癌混合组织cDNA文库,为进一步筛选克隆胃癌的相关癌基因或抑癌基因奠定了基础。

Abstract: Objective: To construct of a lambda phage based cDNA library made from human gastric cancer compound tissue,and to screen and clong gastric cancer related genes.Methods: Total RNA and mRNA was extracted and converted to blunt ended,double stranded cDNA by Oligo（dT） mediated reverse transciption followed by adapter addition Column chromotography isolates residual adapters and small bands Ligation of cDNA to the vector and in vitro packaging of ligated cDNA and introduction into E coliY1090 Titer and amplify the phage.Results: The cDNA library has 3 2×10 ⁶ recombinants,the recombining ratio is 96% The titer of amplified library is 1 2×10 ¹² pfu/L-.Conclusion: The results indicacate that we constructed the human gastric cancer compound tissue cDNA library successfully