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《Journal of Fujian Agriculture and Forestry University(Natural Science Edition)》 2009-02
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Establishment and optimization of ISSR reaction system of Sarcandra glabra

HUANG Yu,RONG Jun-dong,LI Feng-tao,CHEN Li-guang,LI Hong-guang,HE Tian-you,ZHENG Yu-shan(Institute of Industrial Forest,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)  
Taking genomic DNA extracted from Sarcandra glabra(Thunb.) Nakai leaves as the experimental material,the factors which affected the ISSR-PCR amplification such as suitable concentration for dNTPs,dosage for Taq DNA polymerase,the primers,template DNA,annealing temperature and cycles were selected and optimized.The results showed that the 20 μL ISSR reaction system included: 10×PCR Buffer,0.4 mmol·L-1 dNTPs,2U Taq DNA polymerase,0.6 μmol·μL-1 primer,5 ng template DNA.The optimal PCR amplification process was: 2 minutes at 94 ℃ for predenaturation,then followed by 35 cycles,each with 30 seconds at 94 ℃ for denaturation,30 seconds at 44.8 ℃ for annealing,1 minute at 72 ℃ for extension,finally extension at 72 ℃ for 7 minutes and holding the samples at 4 ℃.The system was applied in the amplification of ten varieties of S.glabra,indicating the suitability and stability of the system.
【Fund】: 福建省科技厅重大资助项目(2004YZ02-05);; 福建省中药材GAP工程技术研究中心资助项目(2008Y2001)
【CateGory Index】: S567.239
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