甘薯TD-SSR-PCR反应体系的优化

为探索甘薯最佳TD-SSR-PCR体系,利用L16(43)正交设计研究2×PCR Mix、引物、模板DNA等主要影响因素的适宜浓度,并在此基础上对扩增程序和聚丙烯酰胺凝胶电泳上样量进行优化。结果表明,优化后的TD-SSR-PCR反应体系包括:10μL 2×PCR Mix、100 ng模板DNA、0.4μmol/L引物,1μL甘油,总体积20μL;优化后的扩增程序为:94℃预变性4 min,94℃变性45 s、Tm+5℃~Tm-5℃(每循环退火温度下降0.5℃,Tm选用一对引物中的较小Tm值)退火30 s(退火时间因扩增片段大小而异)、72℃延伸1 min共20个循环,94℃变性45 s、Tm-5℃退火30 s、72℃延伸1 min共15个循环,最后72℃延伸7 min,4℃保存;聚丙烯酰胺电泳上样量以1.5~2μL为宜。在此条件下,利用引物Z37对10份甘薯材料进行PCR扩增,得到的条带清晰、多态性高,表明此条件适用于甘薯的TD-SSR-PCR反应体系。

The Effect of Two Different Hanging Ways on Comprehensive Benefit of Tobacco Leaves Curing

To explore the best TD-SSR-PCR system on sweet potato,an L16 (43) orthogonal design was used to study the suitable concentration of the main influence factors such as 2×PCR Mix,primers and template DNA.And on this basis,the amplification process and the best sample amount of polyacrylamide gel electrophoresis were optimized.The results showed that the optimized 20 μL TD-SSR-PCR reaction system included:10 μL 2×PCR Mix,100 ng template DNA,0.4 μmol/L primers,1 μL glycerol,total volume is 20 μL;The optimized PCR amplification system started at initial denaturation for 4 min at 94 ℃,followed by 20 cycles of denaturation for 45 s at 94 ℃,anneal for 30 s (Annealing time varied depending on the DNA fragment size) at Tm+5 ～Tm-5 ℃ (annealing temperature decreased 0.5 ℃ per cycle,chose the smaller Tm of a pair of primers as the using one),extension for 1 min at 72℃,then 15 cycles of denaturation for 45 s at 94 ℃,anneal for 30 s at Tm-5 ℃,extension for 1 min at 72 ℃,the amplification was completed after extension for 7 min at 72 ℃,finally stored at 4 ℃.The best sample amount of polyacrylamide gel electrophoresis was 1.5-2 μL.Under this condition,clear banding pattern with high polymorphic was obtained by using primer Z37 to amplify 10 sweet potato materials.It indicated that this condition was suitable for TD-SSR-PCR of sweet potato.