HUANG Meng,CHEN Shang-xing,YANG Guang-yao,et al.Optimization of ISSR Amplification Conditions in Ancient Camphor-trees[J].Northern Horticulture,2013,37(07):116-120.
古樟ISSR扩增条件的优化
- Title:
- Optimization of ISSR Amplification Conditions in Ancient Camphor-trees
- 文章编号:
- 1001-0009(2013)07-0116-05
- Keywords:
- ancient camphor-trees; ISSR; reaction condition; optimization
- 分类号:
- S 792.23
- 文献标志码:
- A
- 摘要:
- 采用改良CTAB法提取古樟叶片基因组DNA。利用单因素试验设计，对反应体系中退火温度、模板DNA含量、Mg2+浓度、dNTP浓度、引物浓度、Taq DNA聚合酶用量、去离子甲酰胺等7种因素不同梯度对扩增结果的影响进行了比较分析。结果表明：古樟ISSR-PCR最佳反应体系为，在20 μL PCR反应体积中，含50 ng DNA模板，2.5 mmol/L MgCl2，0.20 mmol/L dNTPS，1 U Taq DNA聚合酶，0.5 μmol/L引物浓度，2%去离子甲酰胺。该反应体系的建立为今后利用ISSR分子技术对古樟的遗传多样性分析提供了技术支撑。
- Abstract:
- The genomic DNA of ancient camphor-trees was extracted from leaves by improved CTAB method.With the design of single factor experiment method，the influences of seven factors including Mg2+，dNTP，DNA content in the template，Taq DNA polymerase，amounts of primer，deionized formamide，and annealing temperature on PCR amplification were analyzed.The results showed that the best ISSR-PCR reaction system of ancient camphor-trees was as follows：50 ng DNA template，2.5 mmol/L MgCl2，0.20 mmol/L dNTPS，1.0 U Taq polymerase，0.5 μmol/L primer and 2% formamide deionized in 20 μL reaction volume.The results would lay a technology foundation for further study of the genetic diversity of ancient camphor-trees with ISSR molecular marking.
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备注/Memo
第一作者简介：黄萌（1989-），女，在读硕士，研究方向为植物系统与演化。E-mail:mengmeng8910@126.com.