淫羊藿嫩叶cDNA文库的构建(英文)

Construction of cDNA library in Epimedium brevicornum leaves

以淫羊藿嫩叶为实验材料,用Trizol方法提取植物总RNA,纯化出mRNA,用SMART（the Switch Mechanism At the5′end of RNA Templates）技术反转录成cDNA,同时使用CHROMA SPIN-400凝胶柱层析纯化cDNA,最后将片断连入λTriplEx2 vector,经包装得到500μL原始文库,文库的滴度为1.2×10^6Pfu/mL。经体内切割后,随机挑选文库的20个阳性克隆进行PCR鉴定,算出文库的重组率为80%,扩增出的片断主要集中在0.5-2kb之间。结果说明文库质量较好,可以用于基因筛选。

The total RNA was extracted from young leaves of Epimedium brevicornum ,and mRNA was purified by oligo （dT）-cellulose column chromatography,which was reverse transcripted into cDNA using SMART （the Switch Mechanism At the 5＇ end of RNA Templates） technique. The resulting cDNA was digested by Sfi,the digested fragments were fractionated by CHROMA SPIN-400,and then ligated to λTriplEx2 vector, the mixture of ligation was finally packed into Lambda virons with packaging extracts. 6 × 10^5 recombinants had been obtained. 80 percent of products were over 500bp. 20 clones were chosen randomly from the library and were screened cDNA inserts using PCR. The results showed that the cDNA library of young leaves of E. brevicornum had been constructed,and it could be used to screening to isolate particular clones.