基因工程实验教学中PCR反应条件的正交优化

Orthogonal Optimization of PCR Experimental System for Escherichia coli DNA in Genetic Engineering Experiment Teaching

采用碱裂解法提取大肠杆菌的质粒DNA,检测提取到的DNA浓度和纯度,并以提取的DNA作为PCR反应体系中的模板,采用搜索性正交设计试验法探求PCR反应体系中4个因素(dNTPs、DNA模板、引物和Taq酶)在5个水平上的最适用量,在此基础上,进一步设计细调性正交试验筛选PCR反应的最适条件。结果表明,在20μl PCR反应体系中,DNA模板5 ng/μl,dNTPs 0.25 mmol/L,引物0.6μmol/L,Taq酶1.5 U/μl,Mg2+浓度1.5 mmol/L时,PCR的效果最好。

The plasmid DNAs of E. coli were extracted by alkaline method in this paper and its concentration and purity were measured and estimated. The scanning orthogonal experimental design was used to explore the PCR amplification system on E. coli in five levels of four fac- tors （dNTPs, DNA template,primer and Taq polymerase）. On the basis of the experimental result, the finely adjusted orthogonal experimental design was performed to optimize three factors at three levels and establish a most suitable PCR system for the plasmid DNAs of E. coil, which contains 0.25 mmol/L dNTPs, 5 ng/μl template, 0.6 μmoL/L primer, 1.5 mmol/L Mg2 ＋ and 1.5 U/μl Taq polymerase in 20 μl reaction system.