摘要
本研究从人睾丸组织的总RNA中纯化mRNA,并以此为模板,在AMV反转录酶的作用下,制备互补的SS-cDNA。用RNaseH降解模板mRNA,经DNA多聚酶Ⅰ作用,合成ds-cDNA。用T_4DNA多聚酶制备cDNA的平末端,在T_4DNA连接酶的作用下,连接Ecorl接头并与λgt11载体重组连接,以大肠杆菌Y1090做受体菌转染,构建人睾丸cDNA文库。结果表明cDNA文库构建成功,将对生殖和避孕进一步研究发挥重要的作用。
In this study, mRNA was isolated from total RNA in human testis. Simple-stranded cDNA was driven by AM.V reverse trancriptase and followed directly by double-stranded cDNA replacement synthesis using RNaseH, DNA polymerase I. After treatment with T_4DNA polymerase to flush the ends and with T_4DNA ligase to add EcoR I adaptors, cDNA molecules was recombined with λgt11 vectors.Human testis cDNA library was constructed by the recombinant phage infected Y1090.The result show that we have successfull constructed human testis cDNA library which is important forstudies reproduction and contraception.
