畜牧与饲料科学 ›› 2010, Vol. 31 ›› Issue (5): 1-1.doi: 10.12160/j.issn.1672-5190.2010.05.002
• 基础科学 • 上一篇 下一篇
易朝辉
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YI Zhao-hui (Xinyang Vocational and Technical College,Xinyang 464000,China)
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摘要: 以豫南黑猪心脏为供试材料,采用改进的异硫氰酸胍法提取心脏总RNA。通过SMART技术反转录合成全长cDNA,经LD-PCR扩增后与pMD18-T载体连接并转化细菌DH5α,测定滴度和重组率,构建成豫南黑猪心脏全长cDNA文库。经初步检测,原始文库的滴度为1.8×10^5cfu/mL,重组率约为94.4%。从原始文库随机挑取13个单菌落过夜培养,提取质粒,经HindⅢ和EcoRⅠ双酶切,凝胶电泳显示插入片段大小在0.3~2.0kb之间。
Abstract: The total RNA from heart tissue of Yunan pig was extracted by isothiocyanate method in the trial.Firstly,full-length cDNAs were synthesized by SMART technique,then a pool of double-stranded cDNA was amplified by LD-PCR technology.The achived cDNA fragments was ligated into pMD18-T vector and transformed into E.coli DH5α to construct the full-length cDNA library.The library was tittered,and the rate of recombination(blue/white) was also determined.The results showed that the primary titer of the constructed cDNA library was 1.8×105 cuf/mL,the recombination rate was about 94.4% .13 transformed germs were selceted and cultured,then their plamids were extracted and identified by restriction enzyme of Hind Ⅲ and EcoRⅠ,the gel electrophoresis showed the length of most cDNA inserted in the library was about 0.3-2.0 kb.
中图分类号:
S828.891
易朝辉. 豫南黑猪心脏cDNA文库的构建与鉴定[J]. 畜牧与饲料科学, 2010, 31(5): 1-1.
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