[Objective] By using the genomic DNA of Cymbidium faberi Rolfe as template,the factors that affect the result of ISSR-PCR reaction system were researched and the optimal system was established.[Method] The genomic DNA...[Objective] By using the genomic DNA of Cymbidium faberi Rolfe as template,the factors that affect the result of ISSR-PCR reaction system were researched and the optimal system was established.[Method] The genomic DNA was extracted from C.faberi Rolfe with method of modified CTAB.Different factors which affected ISSR amplification reaction were optimized.[Result] High-quality genomic DNA was obtained from C.faberi Rolfe.And the optimal reaction system was as follows：25 μl amplification reactions system contained 2.5 μl 10 × PCR buffer,2.0 mmol/L MgCl2,60 ng template DNA,160 μmol/L dNTPs,1.25 U Taq DNA polymerase,0.4 μmol/L ISSR primer and 15.85 μl ddH2O.The optimal amplification procedures were pre-denaturing for 5 min at 94 ℃,followed by 40 cycles of denaturing for 30 s at 94 ℃,annealing for 30 s at a temperature of 2-3 ℃ lower than melting temperature of each primer pair,extension for 50 s at 72 ℃.Then extension step of 7 min at 72 ℃ was performed.[Conclusion] The optimal system could provide a favorable basis for further study on genetic diversity of C.faberi Rolfe by using ISSR molecular marker technique.展开更多
基金Supported by a Program from the State Environmental Protection Administration(20061A0013)~~
文摘[Objective] By using the genomic DNA of Cymbidium faberi Rolfe as template,the factors that affect the result of ISSR-PCR reaction system were researched and the optimal system was established.[Method] The genomic DNA was extracted from C.faberi Rolfe with method of modified CTAB.Different factors which affected ISSR amplification reaction were optimized.[Result] High-quality genomic DNA was obtained from C.faberi Rolfe.And the optimal reaction system was as follows：25 μl amplification reactions system contained 2.5 μl 10 × PCR buffer,2.0 mmol/L MgCl2,60 ng template DNA,160 μmol/L dNTPs,1.25 U Taq DNA polymerase,0.4 μmol/L ISSR primer and 15.85 μl ddH2O.The optimal amplification procedures were pre-denaturing for 5 min at 94 ℃,followed by 40 cycles of denaturing for 30 s at 94 ℃,annealing for 30 s at a temperature of 2-3 ℃ lower than melting temperature of each primer pair,extension for 50 s at 72 ℃.Then extension step of 7 min at 72 ℃ was performed.[Conclusion] The optimal system could provide a favorable basis for further study on genetic diversity of C.faberi Rolfe by using ISSR molecular marker technique.
