均匀设计优化喜盐鸢尾ISSR-PCR体系

以新疆野生喜盐鸢尾DNA为模板,采用U20(54)和U12(34)均匀设计表,通过4因素5水平和4因素3水平两轮均匀优化试验,对影响ISSR-PCR扩增结果的一些因素如Mg2+、dNTP、引物以及TaqDNA聚合酶浓度以及循环数和退火温度等进行优化筛选,建立了适合喜盐鸢尾的最佳ISSR-PCR反应体系:在25μL反应体系中,包括2.5μL 10×PCRBuffer,2.0mmol/L Mg2+,250μmol/L dNTP,0.1μmol/L引物,0.5UTaqDNA聚合酶,40 ng模板DNA。扩增程序为:94℃预变性3 min;接着进行40个循环:94℃变性45 s,52.1℃退火30 s,72℃延伸1.5 min;循环结束后,72℃延伸5 min,4℃保存。

Optimization of ISSR-PCR System of Iris halophila by Uniform Design

U20(54)and U12(34)uniform design diagrams were amplified to optimize the influencing factors of ISSR-PCR system,including Mg2+,dNTP,primer and TaqDNA polymerase.The suitable ISSR-PCR system and ISSR-PCR procedure for Iris halophila of Xinjiang were established as follows:each 25 μL ISSR-PCR amplification reaction solution was consisted of 2.5 μL PCRBuffer,2.0 mmol/L Mg2+,250 μmol/L dNTP,0.1 μmol/L primer,0.5U TaqDNA polymerase and 40ng template DNA.The suitable ISSR-PCR procedure was 1 cycle of denaturing for 3 min at 94℃,40 cycles of denaturing for 45 s at 94℃,annealing for 30 s at 52.1℃,extending for 1.5 min at 72℃ and the last cycle of extending for 5 min at 72℃.