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目的制备用于人KIM-1基因mRNA实时荧光定量PCR检测的标准品质粒。方法通过PCR扩增出目的片断,纯化PCR产物与p MD18-T载体连接,转化宿主菌E.coli DH5α,获得阳性克隆;通过PCR扩增鉴定和测序分析,确认重组质粒完整正确。提取重组质粒测定DNA浓度后,10倍倍比稀释,建立KIM-1质粒标准品。结果 KIM-1基因目的片段成功重组至p MD18-T载体上,扩增目的片段长度为131 bp,测序结果证实所构建的质粒序列与NCBI基因库KIM-1序列性一致。结论成功构建KIM-1基因实时荧光定量PCR标准品质粒。
Objective To prepare a standard plasmid for real-time quantitative PCR detection of human KIM-1 mRNA. Methods The target fragment was amplified by PCR. The purified PCR product was ligated into pMD18-T vector and transformed into E. coli DH5α. The positive clones were obtained by PCR amplification, sequencing and sequencing. The recombinant plasmids were confirmed to be complete and correct. The recombinant plasmid was extracted and the DNA concentration was determined. The KIM-1 plasmid standard was prepared by 10-fold dilution. Results The target fragment of KIM-1 gene was successfully recombined into pMD18-T vector. The length of the target fragment was 131 bp. The sequencing result confirmed that the sequence of the constructed plasmid was consistent with the sequence of KIM-1 in NCBI gene library. Conclusion The KIM-1 gene real-time fluorescence quantitative PCR plasmid was successfully constructed.