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《Guizhou Science》 2007-02
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STUDY ON THE OPTIMIZATION OF RAPD-PCR EXPERIMENTAL SYSTEM IN LIGUSTRUM ROBUSTUM (ROXB) BL

YAN Dong-hai1,ZHENG Dao-jun2,LIANG Yuan-fa 1,LINGHU Chang-di1,LIU Guo-min2,TIAN Yong-hui1(1.The Tea Research Institute of Guizhou Province,Guizhou,Meitan 564100;2.The kudingcha Research Institute of Hainan University,Hainan,Haikou 570228 )  
Using the young buds as the test material,several important factors in RAPD analysis of Ligustrum robustum(Roxb) Bl were systematically studied and optimized,which include the concentration of DNA template,dNTPs,Mg2+ and Taq polymerase,as well as the annealing temperate,the elongation time and the number of thermal cycle.An optimal RAPD-PCR experimental system for L.robustum was established.The optimal experimental conditions were set as the follows: in 25μl reaction system,buffer 2.5μl 10×PCR,Mg2+ 2.5mM,dNTPs 200μm,Taq polymerase 2.0U,random primers 10pmol and DNA template 80ng.The amplification program of the optimized PCR was: At first pre-denaturing at 94℃ for 4min,then 40 cycles of denaturing at 94 ℃ for 30s;annealing at 37 ℃ for 45s;extension at 72 ℃ for 120s;at last extension at 72 ℃ for 10 min.The amplification productions were stored at 4℃.A high reproducibility was obtained under the optimized experimental conditions.
【Fund】: 贵州省自然科学基金资助项目 批准文号:黔科合J字〔2005〕2033号
【CateGory Index】: S571.1
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