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以小菜蛾(Plutella xylostella)基因组DNA为模板,采用L16(45)正交试验设计方法,建立小菜蛾的ISSR最佳反应体系。通过梯度退火试验,确定不同引物的最适退火温度。优化得到的反应体系(20μL)为:Mg2+浓度1.5mmol/L、Taq DNA聚合酶2.5U、dNTPs浓度0.2mmol/L、引物浓度1.25μmol/L、模板DNA量20ng。反应程序为:94℃预变性5.0min;94℃变性45.0s,40～61℃(不同引物退火温度各异)退火1.0min,72℃延伸1.5min,40个循环;72℃延伸10.0min;10℃保存。利用所建立的ISSR-PCR反应体系,获得了清晰、重复性好的DNA谱带。
Based on the genomic DNA of Plutella xylostella, the optimum ISSR reaction system of Plutella xylostella was established by L16 (45) orthogonal design. Through the gradient annealing test, determine the optimal annealing temperature of different primers. The optimized reaction system (20μL) was 1.5mmol / L Mg2 +, 2.5U Taq DNA polymerase, 0.2mmol / L dNTPs, 1.25μmol / L primer and 20ng DNA. The reaction program was as follows: pre-denaturation at 94 ° C for 5.0 min, denaturation at 94 ° C for 45.0 s, annealing at 40-61 ° C for 40 min (annealing temperature varied for different primers) for 1.0 min, extension at 72 ° C for 1.5 min for 40 cycles, extension at 72 ° C for 10.0 min, ℃ save. Using the established ISSR-PCR reaction system, a clear and reproducible DNA band was obtained.