WU Chun-yan,GAO Yi,SONG Ting-yu,et al.Optimization of an ISSR-PCR Reaction System of Chinese Cabbage by Orthogonal Design[J].Northern Horticulture,2012,36(05):127-129.
正交设计优化大白菜ISSR-PCR 反应体系
- Title:
- Optimization of an ISSR-PCR Reaction System of Chinese Cabbage by Orthogonal Design
- 文章编号:
- 1001-0009（2012）05-0127-03
- Keywords:
- Chinese cabbage; ISSR; orthogonal design; PCR reaction system
- 分类号:
- S 634.103.6
- 文献标志码:
- A
- 摘要:
- 以大白菜为试材，采用新型植物基因组DNA提取试剂盒提取大白菜基因组DNA，采用正交实验设计方法，对dNTPs、Mg2+、Taq- DNA聚合酶、引物、及模板 DNA五因素四水平进行优化，筛选并建立了适合大白菜的ISSR-PCR反应体系。结果表明：25 μL的反应体系中含有1.5 mmol/L Mg2+、200 μmol/L dNTP、0.5 UTaq-DNA聚合酶、0.7 μmol/L 引物、30 ng模板DNA。在此基础上探讨了最佳循环次数，应用该优化反应体系，用3个不同循环数对资源DNA进行ISSR-PCR扩增，结果显示优化的反应体系适宜的循环次数是30。
- Abstract:
- Chinese cabbage genomic DNA,extracted from its fresh young leaves by DNA extraction kit was applied to optimize ISSR-PCR amplification system based on orthogonal design.Five factors were considered such as dNTP，Mg2+，Taq- DNA polymerase，primer and template DNA at four levels.A suitable ISSR-PCR reaction system was established.The results showed that 25 μL reaction system containing 1.5 mmol/L Mg2+，200 μmol/L dNTP，0.5 U Taq-DNA polymerase,0.7 μmol/L primer and 30 ng DNA template.The number of cycles was also investigated.Three different cycle numbers of were used to test the optimized reaction system.The results indicated that the appropriate cycle numbers of the optimized reaction was 30.
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备注/Memo
第一作者简介：吴春燕（1978-），女，吉林永吉人，博士，讲师，研究方向为蔬菜种质资源创新与利用。E-mail：cuwu315@163.com。
责任作者：张晓明（1962-），男，硕士，教授，研究方向为蔬菜栽培生理。E-mail：xiaomingzh@126.com。
基金项目：吉林农业大学科研启动基金资助项目（201013）。
收稿日期：2011-12-14