以大豆(Glycine max L.)为材料,研究了PCR反应体系的主要成分对大豆SSR扩增结果的影响,并确定影响SSR扩增结果的各因素的最佳用量.以CTAB法提取的大豆叶片DNA为模板,应用L16(44)正交设计对影响大豆SSR-PCR的主要参数进行优化,建立适合大豆SSR-PCR反应的最佳体系.结果表明:各因素不同水平浓度对PCR反应结果均有显著影响.大豆SSR-PCR优化反应体系为:2.0 μL 10×PCR Buffer,30 ng模板DNA,150μmol/L dNTP,0. μmol/LSSR引物,1.5 U Taq DNA聚合酶,2.0 mmoL/L Mg2+,加ddH2O至终体积20.0μL.优化的PCR扩增程序为:9℃预变性5 min.9℃变性30 s,50℃退火1 min,72℃延伸1 min,共35个循环,72℃延伸5 min,℃保存.同时选用200对大豆引物对2份材料进行扩增,筛选出条带清晰,多态性好的引物7对,用于大豆SSR标记的进一步研究.
The SSR amplified results of soybean was studied to optimize several factors applied in PCR technique system.We made L16(44)orthogonal designs for optimizing the main factors of the SSR2PCR reaction system of Soybean,and the SSR reaction was finally optimized. The results showed that there are significant effects on the results of SSR2PCR under the different levels of every factor,and the optimized SSR-PCR reaction systemwas:2.0μL 10 ×PCR Buffer,30ng DNA template,150μmol/ L dNTP,0.4μmol/ L SSR primer,1.5UTaq DNA polymerase,2. 0 mmol/L Mg2+,adding ddH2O to terminal volume 20.0 μL.The optimized PCR amplification system started at predenaturation for 5 min at 94 ℃,followed by 35 cycles of denaturation for 30 s at 94 ℃,anneal for 1min at 50 ℃,extension for 1 min at 72 ℃,the amplification was completed after extension for 5 min at 72 ℃,then stored at 4 ℃. Seventy2four primer pairs were selected from 200 primer pairs for the SSR analysis based on their reproducible and clear banding patterns,which could be used for further research.
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