猪胰脏细胞cDNA文库的构建

 本文以新鲜猪胰脏为材料,采用异硫氰酸胍法和寡聚(dT)-纤维素亲和层析法,提取了Poly(A)~+RNA,经麦胚无细胞体系鉴定其体外翻译活性,~3H-Leu参入量为空白对照的5倍。参照Gubler和Hoffman等人的方法,以此poly(A~+)RNA为模板,合成总cDNA,并采用多聚物加尾法,与pUC19质粒重组,转化入感受态E.coliJM107,进行分子克隆,其转化率为3.6×10~4克隆子/μgcDNA。并对重组质粒DNA进行了酶切鉴定。

Construction of cDNA Library From mRNA of Porcine Pancreatic Cells

Total RNA was prepared by extraction of porcine pancreas with guanidine thiocyanate.Poly(A+)RNA was selected on oligo(dT)-cellulose.The mRNAs obtained were active in translation as determend in a wheat germ cell-free system.The major part of the mRNAs was identified to be a 18s component by alkaline agarose gel electrophoresis.cDNAs were synthesized according to the method of Gubler and Hoffman with some modifications.The size of the main component was in the range of 0.59-1.97 kb as revealed by agarose gel electrophoresis.The cDNA were then inserted into the pST I site of plasmid pUC19 to form recombinant DNA molecules, which were then introduced to E.coli JM101 and a cDNA library was established with a transformation efficiency of 3.5***********x104 clones/*******ug cDNA.