[Objective] The aim of this study is to establish and optimize the ISSR-PCR system in roughskin sculpin(Trachidermus fasciatus Heckel).[Method] By using the primer of ISSR-63,the concentrations of Taq DNA polymerase,M...[Objective] The aim of this study is to establish and optimize the ISSR-PCR system in roughskin sculpin(Trachidermus fasciatus Heckel).[Method] By using the primer of ISSR-63,the concentrations of Taq DNA polymerase,Mg2+,dNTPs and primer were optimized by orthogonal design for establishing the suitable ISSR-PCR system in roughskin sculpin;moreover,the suitable anneal temperature was yielded from gradient PCR on temperature.[Result]The optimized ISSR-PCR system(20 μl reaction volume)in roughskin sculpin was proved to be:2.5 mmol/L Mg2+,250 μmol/L dNTPs,0.25 μmol/L primer,1 U Taq DNA polymerase,30 ng DNA template and 1×PCR buffer;and suitable anneal temperature was determined to be 50.8 ℃.The established system was further confirmed by using 24 wild roughskin sculpin samples.[Conclusion]The results lay a foundation for the analysis of genetic diversity and germplasm resources of roughskin sculpin.展开更多
With Portulaca oleracea L. as an experimental material, its total DNA was extracted by the improved CTAB method, the ISSR-PCR primers were screened, and the ISSR-PCR reaction system and reaction conditions for P. oler...With Portulaca oleracea L. as an experimental material, its total DNA was extracted by the improved CTAB method, the ISSR-PCR primers were screened, and the ISSR-PCR reaction system and reaction conditions for P. oleracea were Optimized. The results showed that there were 8 primers suitable for ISSR-PCR of P. oleracea. The optimal reaction system had a volume of 25 μl, including 2 x Taq Platinum PCR Master Mix 12.5 μl, primer 2 μl, ddH20 9.5 μl, and DNA template 1μl. The optimized ISSR-PCR of P. oleracea was started with pre-denaturation at 94 ℃ for 360 s, followed by 30 cycles of denaturation at 94 ℃ for 60 s, annealing at 54 ℃ for 60 s and extension at 72 ℃ for 90 s, and completed by extension at 72 ℃ for 300 s.展开更多
以博白大果油茶Camellia gigantocarpa Hu et Huang为材料,通过单因素实验设计和正交设计L18(35)对影响PCR反应体系的主要成分及PCR扩增程序进行优化。建立了稳定的、可重复的博白大果油茶ISSR-PCR扩增反应体系。在20μL的反应体系中,M...以博白大果油茶Camellia gigantocarpa Hu et Huang为材料,通过单因素实验设计和正交设计L18(35)对影响PCR反应体系的主要成分及PCR扩增程序进行优化。建立了稳定的、可重复的博白大果油茶ISSR-PCR扩增反应体系。在20μL的反应体系中,Mg2+浓度为2.5mmol/L,模板DNA用量为20ng,Taq DNA聚合酶为1.75U,dNTPs浓度为0.25mmol/L,引物浓度为0.6mmol/L。反应程序为:94℃预变性4min,94℃变性30s,50℃退火45s,72℃延伸90s,35个循环,72℃延伸7min,4℃保存。展开更多
基金Supported by High Tech Project from Sci-tech Department of Jiangsu Province(BG2006337)~~
文摘[Objective] The aim of this study is to establish and optimize the ISSR-PCR system in roughskin sculpin(Trachidermus fasciatus Heckel).[Method] By using the primer of ISSR-63,the concentrations of Taq DNA polymerase,Mg2+,dNTPs and primer were optimized by orthogonal design for establishing the suitable ISSR-PCR system in roughskin sculpin;moreover,the suitable anneal temperature was yielded from gradient PCR on temperature.[Result]The optimized ISSR-PCR system(20 μl reaction volume)in roughskin sculpin was proved to be:2.5 mmol/L Mg2+,250 μmol/L dNTPs,0.25 μmol/L primer,1 U Taq DNA polymerase,30 ng DNA template and 1×PCR buffer;and suitable anneal temperature was determined to be 50.8 ℃.The established system was further confirmed by using 24 wild roughskin sculpin samples.[Conclusion]The results lay a foundation for the analysis of genetic diversity and germplasm resources of roughskin sculpin.
文摘With Portulaca oleracea L. as an experimental material, its total DNA was extracted by the improved CTAB method, the ISSR-PCR primers were screened, and the ISSR-PCR reaction system and reaction conditions for P. oleracea were Optimized. The results showed that there were 8 primers suitable for ISSR-PCR of P. oleracea. The optimal reaction system had a volume of 25 μl, including 2 x Taq Platinum PCR Master Mix 12.5 μl, primer 2 μl, ddH20 9.5 μl, and DNA template 1μl. The optimized ISSR-PCR of P. oleracea was started with pre-denaturation at 94 ℃ for 360 s, followed by 30 cycles of denaturation at 94 ℃ for 60 s, annealing at 54 ℃ for 60 s and extension at 72 ℃ for 90 s, and completed by extension at 72 ℃ for 300 s.
文摘以博白大果油茶Camellia gigantocarpa Hu et Huang为材料,通过单因素实验设计和正交设计L18(35)对影响PCR反应体系的主要成分及PCR扩增程序进行优化。建立了稳定的、可重复的博白大果油茶ISSR-PCR扩增反应体系。在20μL的反应体系中,Mg2+浓度为2.5mmol/L,模板DNA用量为20ng,Taq DNA聚合酶为1.75U,dNTPs浓度为0.25mmol/L,引物浓度为0.6mmol/L。反应程序为:94℃预变性4min,94℃变性30s,50℃退火45s,72℃延伸90s,35个循环,72℃延伸7min,4℃保存。
