摘要
目的:构建莱芜猪肝脏组织全长cDNA文库,以便研究与莱芜猪优良性状相关的基因。方法:采用改良的异硫氰酸酸胍一步法制备总RNA;利用SMART技术,以PrimeScript反转录酶逆转录合成第一链cDNA,通过LD-PCR扩增获得cDNA双链;经蛋白酶K消化和CHROMA SPIN-400柱分级分离后,收集500 bp以上的cDNA片段,并与pMD18-T载体连接,转化大肠杆菌DH5α感受态细胞,建成原始文库;随机挑取单菌落,用HindⅢ和EcoRⅠ进行双酶切鉴定重组子插入片段大小。结果:经鉴定,原始文库的滴度为2.8×105 cfu/mL,重组率约为98%,插入片段大小为0.5～2 kb,平均插入片段长度大于1 kb。结论:建立的cDNA文库质量良好,可以用于目的基因的筛选。
Objective：To construct a full-length cDNA library of Laiwu pig liver tissue for reserach on the genes related to the good characteristics of Laiwu pig.Methods：Total RNA was extracted by using a modified one-step acid guanidinium thiocyanate method.The first-strand cDNA was synthesized through reverse transcription using PrimeScript reverse transcriptase and the long-distance PCR（LD-PCR） assay was used to amplify double-strand cDNA based on the SMART（switching mechanism at 5′ end of RNA transcript） technique.After digestion by proteinase K and size fractionation using CHROMA SPIN-400 columns,the cDNA fragments longer than 500 bp were ligated to pMD18-T vector.The ligation mixture was transformed into the competent cells of E.coli DH5α to complete the construction of the unamplifed cDNA library.Total 36 single clones were picked randomly from the library for screening the size of cDNA inserts through double digestion with HindⅢ and EcoRⅠ.Results：By e-valuation,the titer of unamplifed cDNA library was estimated as 2.8×105 cfu / mL with the percentage of recombi-nant clones about 98%.The insert sizes ranged from 0.5 kb to 2 kb with the average length larger than 1 kb.Conclusion：The results showed that the quality of this library is good enough for further cloning and expressing target genes.
出处
《生物技术通讯》
CAS
2010年第3期397-400,共4页
Letters in Biotechnology
基金
国家科技基础条件平台建设项目(2005DKA21101-07)
关键词
莱芜猪
肝脏
CDNA文库
SMART技术
Laiwu pig
liver
cDNA library
switching mechanism at 5′ end of RNA transcript technique
