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用Tripure试剂盒提取斜带石斑白细胞总RNA ,反转录合成第一链cDNA ,进行长距离PCR ,蛋白酶K消化 ,SfiI酶切 ,通过CHROMASPIN - 40 0柱 ,回收大于 5 0 0bp的cDNA ,与λTripIEx2载体连接 ,体外包装后构建了斜带石斑细胞的cDNA文库。库容 1 .6 5× 1 0 6 ,重组频率 82 % ,扩增后滴度 7.5× 1 0 9pfuml- 1。插入片断长度 5 0 0～ 2 30 0bp ,最多的是在 75 0～ 1 0 0 0bp范围。本文库可作为筛选斜带石斑细胞因子基因的重要资源
Total RNA was extracted from the stained lenticule leukocytes by Tripure kit and the first strand cDNA was reverse transcribed. The cDNA was amplified by PCR, digestion with Proteinase K and digested with SfiI. The cDNA was recovered by CHROMASPIN - 40 0 column, Connected with λTripIEx2 vector, and packaged a cDNA library of stained lamellar cells in vitro. The storage capacity was 1.56 × 106, the recombination frequency was 82%, and the titer after the amplification was 7.5 × 109pfum1-1. Insert fragment length of 500 ~ 2 30 0bp, the most in the 75 0 ~ 100bp range. This library can be used as an important resource for screening the gene of serpin