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Study on ISSR-PCR reaction system for Clintonia udensis Trautv.et Mey.

WANG Yi-ling~(1,2),LIAO Hai-min~(1,3),ZHAO Gui-fang~1(1.College of Life Science,Northwest University,Shanxi Xian 710069,China;2.College of Life Science,Shanxi Normal University,Shanxi Linfen 041004,China;3.College of Life Science,Guizhou University,Guizhou Guiyang 550025,China)  
A suitable ISSR-PCR amplification system of Clintonia udensis Trautv.et Mey.was established and the annealing temperature of primers was affirmed.Orthogonal design was used to optimize the reaction condition of ISSR experiment in four factors(Mg~(2+),dNTP,Taq polymerase dosage,primer) at four levels respectively.Based on T_m-5℃±5℃ to(design)a temperature grades,the annealing temperature of different primer was tested.For 873,its annealing temperature was 53℃.Different factors' combination on different level was tested for different primers,the optimal combination system of different primers was found.And the tenth combination of primer 897 was the best(3.0 mmol·L~(-1)Mg~(2+);0.2mmol·L~(-1)dNTP;0.1μL Taq DNA polymerase dosage;0.2μmol·L~(-1)primer concentration).PCR reaction volume of 10μL,2.5-3.0mmol·L~(-1)Mg~(2+),0.15-0.20mmol·L~(-1)dNTP,0.20-0.25μmol·L~(-1)primer concentration,0.1μL Taq DNA polymerase dosage,50ng template DNA.And proper annealing temperature was 48-62℃.
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