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Establishment and optimization of the ISSR-PCR reaction system in Stipa krylovii

SUI Xiao-qing,WANG Kun (Institute of Grassland Science,College of Animal Science and Technology,China Agriculture University,Beijing 100094,China)  
An orthogonal design combined with single factor experiment was applied to optimize ISSR(inter-simple sequence repeat)-PCR(polymerase chain reaction) amplification system of Stipa krylovii.The effect of the five main reaction system elements(Taq DNA polymerase,Mg2+,DNA template,dNTP and primer) on ISSR-PCR were tested.A suitable ISSR-PCR reaction system(25 μL) for S.krylovii contains 50 ng template DNA,1.5 mmol/L Mg2+,0.2 mmol/L dNTP,0.8 μmol/L primer,1 U Taq DNA polymerase and 10×buffer 2.5 μL.This will provide the basis for the analysis of diversity,map construction and gene localization in S.krylovii with ISSR markers.
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