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《Biotechnology》 2009-06
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Establishment of ISSR Reaction System of Trichosanthes kirilowii Maxim

LI Hui-hui,ZHU Yu-qiu,SI Jin-pin,GAO Yan-hui(Lab of Biotechnology,Zhejiang Forestry College,Lin'an 311300,China)  
Objective:The optimal orthogonal ISSR-PCR of Trichosanthes lay a foundation for Trichosanthes ISSR molecular marker technology in this study.Method:The optimal experiments of the importmant parameters(DNA template,MgCl2,dNTPs,primers,TaqDNA polymerase) of Trichosanthes ISSR-PCR amplification through the orthogonal experimental design method,and different PCR temperatures,were studied.Result:The suitable ISSR-PCR reaction system(20μl) was established as follows:30ng DNA templet,2.0mmol·L-1 Mg2+,0.3mmol·L-1 dNTPs,0.5U TaqDNA polymerase,0.5μmol·L-1 primer;the suitable PCR reactions were predenaturing at 94℃for 5min,35 cycles of denaturation at 94℃ for 30s,annealing at 52℃-55℃ for 1 minute and extension at 72℃ for 2 minutes,with 7 min final extension at 72℃,and then saved at 4℃.Conlucsion: It provided a more objective and more reliable method of the best Trichosanthes ISSR reaction system for identification Trichosanthes germplasm.
【Fund】: 浙江省科技厅项目(2008C32012)资助
【CateGory Index】: S567.239
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