摘要
从正常培养的SH-SY5Y细胞中提取总RNA,经oligo(dT)纤维素柱纯化分离出mRNA,然后以oligo(dT18)为锚定引物反转录生成单链cDNA再以此为模板合成DNA的第二条链;将双链DNA经Sau3AI酶切之后,接上接头,经通用引物和选择性引物进行扩增;采用5%非变性PAGE胶电泳分离后,用银染的方法显示DNA条带;在直视下回收DNA带,经过扩增后克隆入pMD18-T载体中并鉴定。结果建立了非放射性同位素的银染RD-PCR方法,用于分离基因片段。
To developrestrictiondisplaypolymerasechainreaction(RD-PCR)withsilver-staining,isolatethegenefrag-mentsfromSH-SY5Ycellline.mRNAwas extractedfromSH-SY5Ycells.ThesinglestrandcDNAwas synthesizedusing an anchoredprimeroligo(dT18),thenthesecondstrandwasyieldedby nick-translation.Thedoublestrandswerecleftwith restrictionenzymeSau3AI,thenthefragmentswereligatedwithan adapter.Theproductof ligationwas amplifiedwiththe universalprimerandselectedprimers.TheDNAbandswerestainedwithsilver,thenrecoveredfromnon-denaturinggeland remplifiedby PCRusingtheselectedprimers.Theproductsof RCRwereclonedintothepMD18-Tvector.Somegenefrag-mentswereclonedby methodof silver-stainingRD-PCR.TheRD-PCRthchniquecombinedwithsilver-stainingis a useful,simple,efficientmethodforisolatingthegenefragmentsfromcells.
出处
《生物技术通讯》
CAS
2002年第1期42-44,共3页
Letters in Biotechnology
关键词
银染色法
RD-PCR
基因层断
分离
silver-staining
restrictiondisplaypolymerasechainreaction(RD-PCR)
