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首页> 外文期刊>Journal of Molecular Genetics >Optimizing System of SSR-PCR in Pinus radiata and Pinus tabulaeformis
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Optimizing System of SSR-PCR in Pinus radiata and Pinus tabulaeformis

机译：辐射松和油松SSR-PCR优化体系

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Reaction system of SSR-PCR was optimized by orthogonal design using 21 Pinus radiate and 3 Pinus tabulaeformis originated from the different locations. The results indicated that the optimal reaction system of SSR for P. radiata and P. tabulaeformis were described as follows each DNA amplification was carried out in a volume of 25 μL containing 0.3 U Taq polymerase, 1.5 μL Mgcl2 (2.0 mmol L-1), 160-200 ng genomic DNA, 0.3 μL each of SSR forward and reverse primers (0.6 μmol L-1) and 2.0 μL dNTPs (0.2 mmol L-1). DNA amplification for SSR was performed for initial 3 min at 94°C for pre-denaturing, followed by 30 cycles at 94°C for 30 sec for denaturing, 45 sec at 45-49°C for annealing, 30 sec at 72°C for extension. The reaction was terminated with 5 min extension at 72°C. The reaction system was verified by using 24 materials. The numbers of alleles per primer were 5-10 and the range of polymorphic bands was 100-250 bp.The results suggested that the reaction system described as above exhibited high polymorphic percentage and good stability and repetition.
机译：通过正交设计优化了SSR-PCR的反应体系，使用了21个辐射松和3个来自不同部位的油松。结果表明，针对S. radiata和P. tabulaeformis的SSR最佳反应体系描述如下，每个DNA扩增均在25μL的体积中进行，其中包含0.3 U Taq聚合酶，1.5μLMgcl2（2.0 mmol L-1） ，160-200 ng基因组DNA，每个SSR正向和反向引物（0.6μmolL-1）和2.0μLdNTPs（0.2 mmol L-1）各0.3μL。用于SSR的DNA扩增在94°C进行最初的3分钟以进行预变性，然后在94°C进行30个循环以进行变性30秒，在45-49°C进行退火以进行45秒退火，在72°C进行30秒扩展。在72℃延伸5分钟终止反应。通过使用24种材料验证了反应体系。每个引物的等位基因数目为5-10，多态性条带范围为100-250 bp。结果表明，上述反应体系具有较高的多态性百分比，并且具有良好的稳定性和重复性。

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