An one factor test was used to optimize ISSR-PCR amplification system on macadamia in four levels of five factors(Taq DNA polymerase,template DNA,dNTPs,primer and Mg 2+,respectively) in this study.The results showed t...An one factor test was used to optimize ISSR-PCR amplification system on macadamia in four levels of five factors(Taq DNA polymerase,template DNA,dNTPs,primer and Mg 2+,respectively) in this study.The results showed that the 25 μL reaction system consisted of 1×PCR buffer,1 U Taq DNA polymerase,20 ng template DNA,0.15 mmol·L-1 dNTPs,0.25 μmol·L-1 primer and 2.5 mmol·L-1 Mg 2+.In addition,adding 0.4% formamide was able to reduce the background noise.The optimal PCR amplification process was as the following:1 cycle initial denaturalization at 94 ℃ for 5 min,followed by 35 cycles,which included denaturalization at 94 ℃ for 30 s,annealing for 1 min,and extension at 72 ℃ for 2 min,and then extension at 72 ℃ for 7 min,and finally holding the samples at 4 ℃.展开更多
文摘An one factor test was used to optimize ISSR-PCR amplification system on macadamia in four levels of five factors(Taq DNA polymerase,template DNA,dNTPs,primer and Mg 2+,respectively) in this study.The results showed that the 25 μL reaction system consisted of 1×PCR buffer,1 U Taq DNA polymerase,20 ng template DNA,0.15 mmol·L-1 dNTPs,0.25 μmol·L-1 primer and 2.5 mmol·L-1 Mg 2+.In addition,adding 0.4% formamide was able to reduce the background noise.The optimal PCR amplification process was as the following:1 cycle initial denaturalization at 94 ℃ for 5 min,followed by 35 cycles,which included denaturalization at 94 ℃ for 30 s,annealing for 1 min,and extension at 72 ℃ for 2 min,and then extension at 72 ℃ for 7 min,and finally holding the samples at 4 ℃.
