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Study on Optimization for ISSR Reaction System of Peony with Orthogonal Design

WANG Jia et al(College of Landscape Architecture,Beijing Forestry University,Beijing 100083)  
With Paeonia ostii genomic DNA as a template,the orthogonal design was used to optimize ISSR amplification system of Peony.A suitable ISSR reaction system was established,namely 10 μl reaction system containing 1×PCR buffer,2.5 mmol/L Mg2+,0.4 mmol/L dNTPs,1UTaq DNA polymerase,0.75 μmol/L primer,10~20 ng DNA templet.PCR reactions were pre-denaturing at 94 ℃ for 3 min,35 cycles of de-naturation at 94 ℃ for 45 s,annealing at 45~60 ℃ for 45 s and extension at 72 ℃ for 90 s,with a 7 min final extension at 72 ℃,and then saved at 4 ℃.The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR.Each primer has different annealing temperature.
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