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以宁夏枸杞叶片为材料,采用异硫氰酸胍-酚-氯仿法提取完整的总RNA,利用PolyATract mRNA I-solation System(Promega公司)分离mRNA,然后利用Universal RibocloneRcDNA Synthesis System(Pro-mega公司)合成双链cDNA,在cDNA末端连接上EcoRⅠ接头,并与λExCell载体连接,利用PackageneLambda DNA Packaging System进行包装,构建出滴度为2.78×105pfu/mL,重组效率为88.1%的枸杞叶片cDNA文库。用Digoxigenin(DIG)标记龙胆草IPI探针,并对枸杞叶片cDNA文库进行筛选,获得7个IPIcDNA阳性克隆。释放质粒后,进行酶切鉴定,IPI阳性克隆cDNA插入片段的大小为1.5 kb左右。
The whole RNA was extracted by guanidine isothiocyanate-phenol-chloroform method using Lycium barbarum leaves as material, mRNA was isolated by PolyATract mRNA I-solation System (Promega), and then amplified by Universal Riboclone R cDNA Synthesis System (Pro-mega) Double-stranded cDNA was synthesized. EcoRⅠ adapter was ligated to the cDNA ends and ligated with λExCell vector. The package was packaged by Lambda DNA Packaging System to construct a cDNA library of Lycium barbarum with a titer of 2.78 × 105pfu / mL and a recombination efficiency of 88.1%. The gentian IPI probe was labeled with Digoxigenin (DIG), and the cDNA library of Lycium barbarum leaves was screened to obtain 7 IPIcDNA positive clones. After the plasmid was released and digested with restriction endonucleases, the size of the IPI-positive cloned cDNA insert was about 1.5 kb.