单头赤眼蜂DNA提取方法比较及其PCR反应体系优化

摘要

The CTAB method,SDS method,Chelex method and Acry1 Carrier-CTAB method for DNA extraction was compared using single-head Trichogramma, and PCR reaction of the 5 factors (Mg2+, dNTPs, primer, DNA template, Taq polymerase) 4 leveles screening was done using L16 (45) orthogonal test for extracted Trichogramma genomic DNA. The results showed that the Acryl Carrier-CTAB method was better than the other three methods, and adding Acryl Carrier could significantly enhanced the effects of DNA extraction . We made orthogonal designs for optimizing the PCR system. The optimal reaction system of PCR (25mL) was 1.50mmol·L-1 Mg2+, 0.25m mol·L-1 dNTPs, 0.30μmol·L-1 primer, 2.00ul DNA templates, 2.00U Taq DNA polymerase.%为了探明有效的基因组DNA提取方法和PCR反应体系,从而得到符合分子生物学实验要求的基因组DNA模板的数量和质量,以单头松毛虫赤眼蜂为材料,应用CTAB法、SDS法和Chelex法进行基因组DNA提取效果的比较,并将核酸助沉剂用于赤眼蜂DNA的提取中,然后将提取的基因组DNA采用L16(45)正交设计试验,对影响赤眼蜂基因组DNA PCR反应体系的5因素(Mg2+浓度、dNTPs浓度、引物浓度、DNA模板、Ta聚合酶)4水平进行筛选.结果表明:CTAB法优于SDS法和Chelex法,且核酸助沉剂的加入明显增强了模板DNA提取效果.同时确立的ITS2的PCR优化反应体系为:总体积为25mL,Mg2+浓度为1.50mM,dNTPs浓度为0.25mM,引物浓度为0.30μM,DNA模板取2.00μL,Taq聚合酶量为2.00U.