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为克隆人肝癌相关基因，本实验从提取人肝癌细胞总ＲＮＡ入手，分离其ｍＲＮＡ。以ＮｏｔＩ／ｏｌｉｇｏ（ｄＴ）１２～１８为引物合成双链ｃＤＮＡ，除去小于４００ｂｐ的ｃＤＮＡ片段后，连接ＥｃｏＲⅠ人工接头，经ＮｏｔⅠ酶酶切，插入定向克隆表达载体λＥｘｃｅｌＮｏｔⅠ／ＥｃｏＲⅠ／ＣＩＰ，体外包装后转染大肠杆菌ＮＭ５２２，构建了人肝癌细胞ＢＥＬ－７４０２定向克隆表达ｃＤＮＡ文库。经鉴定，文库含５×１０５个重组子，重组率为９５．２％，文库中插入ｃＤＮＡ的平均大小为１ｋｂ。说明该文库适于筛选各种丰度ｍＲＮＡ的ｃＤＮＡ克隆。
For the cloning of human liver cancer related genes, this experiment started by extracting human hepatoma cell total RNA and separating its mRNA. Double-stranded cDNAs were synthesized using NotI/oligo(dT) 12-18 as primers, cDNA fragments smaller than 400 bp were removed, and EcoRI artificial linkers were ligated, digested with NotI enzyme, and inserted into the directional expression vector λExcelNotI/EcoRI/CIP, after in vitro packaging. The E. coli NM522 was transfected, and a directional cloned cDNA library of human hepatocellular carcinoma BEL-7402 was constructed. After identification, the library contained 5×105 recombinants, the recombination rate was 95.2%, and the average size of the inserted cDNA in the library was 1 kb. This library is suitable for screening cDNA clones of various abundance mRNAs.