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《Pratacultural Science》 2011-05
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Optimization of the ISSR reaction system of Elymus nutans by orthogonal design

LIU Xin-liang1,2,DE Ying1,ZHAO Lai-xi1(1.Grassland Research Institute,Chinese Academy of Agricultural Science,Inner Mongolia Hohhot 010010,China;2.Graduate School,Chinese Academy of Agricultural Science,Beijing 100081,China)  
Five factors(DNA polymerase,Mg2+,template DNA,dNTPs,primer) in ISSR-PCR reaction system were carried on the optimized experiment by taking the genomic DNA of Elymus nutans as template.The optimum reaction system and reaction process of E.nutans ISSR-PCR analysis were established.The results of this study showed that the volume of reaction system was 25 μL.It included 2.5 μL 10×buffer(except Mg2+),1.0 U TapDNA polymerase,2.0 mmol/L Mg2+,30-120 ng template DNA,0.25 mmol/L dNTP and 0.25 μmol/L primer.Eleven primers with stable amplification and rich polymorphism were obatined based on the optimal PCR reaction system.The reaction program was devised for 2 min at 94℃ in the first circle,1 min at 94℃,51℃ for 1 min(depended on different primer),72℃ for 1.5 min for 41 cycles,and 72℃ for 10 min for extending,which was obatined across the gradient PCR experiments,including temperature gradient,cycle gradient and extending time gradient experiments.Validation of the optimistic ISSR-PCR reaction system and amplification program showed that the reaction system and amplification program had high stability and good reproducibility.
【Fund】: 2008年度中央级公益性科研院所基本科研业务费专项资金(中国农业科学院草原研究所 2008-Z-1);; 农业部“948”牧草种质资源收集与创新利用平台建设(2006-G8(4)-21);; 国家科技基础条件平台建设项目(2005DKA21007)
【CateGory Index】: S543.9
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