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通过采用L16(45)正交设计试验设计对影响SSR-PCR的Taq聚合酶用量、Mg2+浓度、DNA模板浓度、dNTP浓度和引物浓度等5个因素在4水平上进行筛选,建立了适宜宽翅曲背蝗(Pararcyplera microptera meridionalis Ikonni-kov)的SSR反应体系和扩增程序,优化后的检测体系更为经济有效。宽翅曲背蝗SSR-PCR的优化体系总体积为20μl,包括:Taq聚合酶为1.5U/20μl、Mg2+浓度为1.5mmol/L、dNTPs浓度为0.2mmol/L、引物浓度为0.2μmol/L及DNA模板为180ng/20μl。PCR适宜扩增程序为:94℃预变性5min,94℃变性45 s,55℃退火45s,72℃延伸45 s,35次循环,最后72℃延伸8min。
Five factors influencing SSR-PCR such as Taq polymerase, Mg2 + concentration, DNA template concentration, dNTP concentration and primer concentration were screened at 4 levels using L16 (45) orthogonal design. The SSR reaction system and amplification program of Pararcyplera microptera meridionalis Ikonni-kov, the optimized detection system is more cost-effective. The total volume of SSR-PCR optimized system was 20 μl, including 1.5 U / 20 μl Taq polymerase, 1.5 mmol / L Mg2 +, 0.2 mmol / L dNTPs and 0.2 μmol / L And DNA template was 180 ng / 20 μl. PCR suitable amplification program: 94 ℃ denaturation 5min, 94 ℃ denaturation 45s, 55 ℃ annealing 45s, 72 ℃ extension 45s, 35 cycles, the last 72 ℃ extended 8min.