摘要
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 ℃.
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 x 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 ℃.
出处
《林业科学》
CAS
CSCD
北大核心
2008年第5期160-164,共5页
Scientia Silvae Sinicae
基金
农业科技成果转化资金项目(04EFN216900382)
农业结构调整重大技术研究专项(04-08-02A)
