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大鼠肌细胞生成素基因的克隆

Molecular cloning of rat myogenin gene

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【作者】 姜浩徐建光刘靖波顾玉东李继峰胡韶楠

【Author】 JIANG Hao, XU Jian guang, LU Jing bo, et al. Department of Hand Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China

【机构】 复旦大学华山医院手外科复旦大学华山医院手外科 200040上海200040上海

【摘要】 目的 设计特异引物克隆大鼠肌细胞生成素 (myogenin)的cDNA。方法 用逆转录聚合酶链反应 (reversetranscriptionpolymerasechainreaction ,RT PCR)合成myogenincDNA ;经提纯后 ,连接到克隆载体pGEM T ,形成重组的克隆载体pGEMT myogenin。pGEMT myogenin转化大肠杆菌Ecoli DH5α内扩增 ;提取、纯化的pGEMT myogenin ,经酶切、PCR鉴定及DNA测序 ,来判断所克隆myogenincDNA的正确性。结果 RT PCR产物经凝胶电泳鉴定 ,含有大小正确的DNA片段 ;提取、纯化的pGEMT myogenin质粒经限制性内切酶酶切、PCR鉴定 ,含有大小正确的DNA片段 ,且该片段含有所设计的酶切位点。DNA测序结果证实所克隆的myogenincDNA序列正确。结论 该研究成功地克隆了myogenincDNA。

【Abstract】 Objective To clone rat myogenin cDNA by design of specific primers.Methods The myogenin cDNA was composed by reverse transcription polymerase chain reaction (RT PCR).The purified products were ligated with the cloning vecter pGEM T(Promega, USA). The recombinant cloning vector pGEMT myogenin was formed. Then pGEMT moygenin was transformed into Escherichia Coli DH5α for amplifying target gene. After plasmids pGEMT myogenin were extracted and purified, the coding sequence of myogenin cDNA was verified by digestion with restriction endonuclease, PCR checkup and DNA sequencing. Results The RT PCR products was checked by agarose gel electrophoresis analysis. The result confirmed the right length of inserted DNA fragment. The extracted and purified plasmids pGEMT myogenin were digested by restriction endonuclease and checked up by PCR. The result confirmed the right length of inserted DNA fragment with designed digested site. The DNA sequencing result confirmed the cloned myogenin cDNA sequence was right. Conclusion The myogenin cDNA is cloned successfully.

【基金】 国家 973创伤基础研究基金资助项目 (G1 9990 542 0 6);复旦大学Med X基金;复旦大学创新基金资助项目 (EXF0 0 0 30 1 )
  • 【文献出处】 中华手外科杂志 ,Chinese Journal of Hand Surgery , 编辑部邮箱 ,2003年01期
  • 【分类号】R346
  • 【被引频次】5
  • 【下载频次】67
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