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目的:设计并制备65bp-500bp范围的荧光标记DNA分子量内标。方法:p MD18-T Vector连接任意一段割胶回收的DNA片段,克隆后提取重组质粒经双酶切后作为模板,在其上游设计一条固定引物并用荧光标记,在其下游设计一系列引物,经PCR扩增后在ABI 3100遗传分析仪上检测。结果:扩增产物DNA片段大小分别为65bp、105bp、149bp、200bp、241bp、269bp、311bp、345bp、400bp、450bp、500bp,与预期片段大小一致。结论:11个片段经混合调平后,峰型良好,出峰位置均匀,可用于毛细管电泳中DNA片段大小的确定。
OBJECTIVE: To design and prepare a 65 bp-500 bp internal standard fluorescent-labeled DNA. METHODS: The pD18-T Vector was used to ligate any fragment of DNA recovered by tapping. After cloning, the recombinant plasmid was digested with restriction enzyme and used as a template. A fixed primer was designed upstream of the vector and fluorescently labeled. A series of primers were designed downstream. After amplification, the ABI 3100 Genetic Analyzer was used. Results: The sizes of DNA fragments were 65bp, 105bp, 149bp, 200bp, 241bp, 269bp, 311bp, 345bp, 400bp, 450bp and 500bp, respectively. Conclusion: The 11 fragments were mixed and leveled, the peak shape is good, the peak position is uniform, which can be used for the determination of DNA fragment size in capillary electrophoresis.