木质素合成酶COMT基因的PCR扩增条件优化

Optimization of PCR Conditions for Amplifying the Lignin Biosynthesis COMT Gene

以2×CTAB法从欧美杨107号嫩叶提取的基因组DNA为试材,建立了木质素合成酶COMT基因的PCR扩增体系。通过对PCR反应的影响因子——模板DNA用量、dNTP浓度、DNA聚合酶量、引物浓度和退火温度进行优化,最终建立了扩增COMT的PCR优化体系为:在20μL的反应体系中,dNTP为2.5 pmol,引物各为5 pmol,模板为5 ng,Red TaqTMDNA聚合酶1.25 U,退火温度为51℃,可以扩增出清晰的扩增带。

The lignin biosynthesis enzyme COMT gene was amplified from poplar （Populus× eurarnericana cv. 74/76） leaves. The PCR amplification conditions were optimized by adjusting the contents of DNA template,dNTP,Taq DNA polymerase,primers and melting temperature. The optimal reaction system of PCR was as follows： 2μl 10 × PCR buffer, 1.25U Red Taq^TM DNA polymerase,5 ng DNA templates,5pmol primers,2. 5pmol dNTP mixtures in a total 20μL reaction volume with the melting temperature at 51℃.