单头赤眼蜂DNA提取方法比较及其PCR反应体系优化

DNA Extraction of Single-head Trichogramma and PCR System Optimization

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、Taq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。

The CTAB method,SDS method,Chelex method and Acryl 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.