知识与财富的链接
【目的】探寻控制羊毛性状的相关基因,构建了新吉细毛羊皮肤组织全长cDNA文库。【方法】采用Clontech公司的CreaterTM SMARTTM cDNA library construction kit,用Biozol试剂提取新吉细毛羊皮肤组织总RNA后,以反转录酶SuperscriptTMⅡ反转录为第一链cDNA,然后通过LD-PCR合成并扩增ds cDNA,扩增产物经纯化、SfiⅠ酶切、过CHROMA SPIN-400柱去除小片段后,连接到SfiⅠ消化过的pDNR-LIB(带有SfiⅠA和B 2个位点)质粒载体中,最后用热击转化法将重组质粒转化到E.coliDH5α内,得到新吉细毛羊皮肤组织全长cDNA原始文库,扩增后的文库分装后用50 mL离心管保存。【结果】构建的cDNA文库约含4.2×105个独立克隆,重组效率为100%,插入片段多为0.5~3.0 kb,平均插入片段长度为1.3 kb,符合建库要求。【结论】经检测所构建的文库库容量满足基因筛选要求,可用于特异表达基因全长序列。
【Objective】 The study was to construct a long length cDNA library of skin tissue for Xinji Fine wool Sheep to seek for genes related to wool trait.【Method】 The library was constructed by SMART technique using CreaterTM SMARTTM cDNA library construction kit.The total RNA was extracted from sheep skin tissue using Biozol isolation reagent.Single strand cDNA was synthesized using SuperscriptTM Ⅱ reverse transcriptase,and double strand cDNA was synthesized and amplified by long distance PCR.The PCR products were purified by QIAquickTM PCR Purification kit.After digestion with SfiⅠand size fractionation using CHROMA SPIN-400 columns,ds cDNA was ligated to the SfiⅠdigested,dephosphorylated pDNR-LIB vector.The ligation mixture was transformed into E.coli DH5α by thermal shock.【Result】 The cDNA library contained 4.2×105 independent clones with DNA inserts of 0.5-3.0 kb.The recombination rate was 100% and the average length of inserts was 1.3 kb.【conclusion】 The capacity of this library can meet the requirement for gene screening and provide a foundation to isolate specific genes in skin for Xinji Fine wool Sheep.