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白桦ISSR-PCR反应体系的优化
Optimization of ISSR-PCR Reaction System for Betula platyphylla Suk
【摘要】 以白桦基因组DNA为模板,研究了ISSR的影响因素,建立一套适宜于白桦的ISSR优化反应体系及程序。即20μL反应体系中,Mg2+浓度范围为0.5~3.0mmol·L-1,dNTPs浓度范围为0.10~0.30mmol·L-1,Taq聚合酶浓度范围为1.0~2.0U,模板DNA浓度为30~120ng,同时含有RNA的模板DNA对ISSR扩增也略有影响,引物浓度范围为0.2~0.4μmmol·L-1。通过梯度PCR测验,确定了适宜的退火温度为49℃。反应程序为:第一个循环基因组DNA经94℃预变性5min;30个循环为94℃变性30s,49℃退火30s,72℃延伸30s;最后一个循环完成后,在72℃延伸7min。
【Abstract】 Using the genomic DNA extracted from Betula as template, the factors influenced ISSR were discussed. A set of ISSR optimized reaction systems and programs were set up. Results showed that for a reaction system with the volume of 20μL, the optimal parameters were obtainedas Mg~ 2+ concentration for ISSR 1.5~3.0mmol·L~ -1 , and the suitable dNTPs concentration 0.10~0.25mmol·L~ -1 , proper quantity of Taq polymerase 0.5~2.0U, appropriate quantity for template DNA 30~100ng, and the concentration of random primer 0.2~0.4μmmol·L~ -1 . The reaction program was devised for 5 minutes of predenaturalization at 94℃, 30 cycles of 30 seconds for denaturalization at 94℃, 30 seconds of anneal at 49℃, 30 seconds of extension at 72℃, and 7minutes of extension at 72℃ in the final cycle.
- 【文献出处】 东北林业大学学报 ,Journal of Northeast Forestry University , 编辑部邮箱 ,2005年06期
- 【分类号】S792.153
- 【被引频次】53
- 【下载频次】272