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纸质出版日期:2015
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杨妮, 苏伟敏, 靳雅惠, 等. 适用于SSR分析的广西莪术DNA提取方法考察[J]. 中国实验方剂学杂志, 2015,21(4):80-83.
YANG Ni, SU Wei-min, JIN Ya-hui, et al. Investigation of Extracton Methods of DNA from for Simple Sequence Repeat Analysis[J]. Chinese journal of experimental traditional medical formulae, 2015, 21(4): 80-83.
杨妮, 苏伟敏, 靳雅惠, 等. 适用于SSR分析的广西莪术DNA提取方法考察[J]. 中国实验方剂学杂志, 2015,21(4):80-83. DOI: 10.13422/j.cnki.syfjx.2015040080.
YANG Ni, SU Wei-min, JIN Ya-hui, et al. Investigation of Extracton Methods of DNA from for Simple Sequence Repeat Analysis[J]. Chinese journal of experimental traditional medical formulae, 2015, 21(4): 80-83. DOI: 10.13422/j.cnki.syfjx.2015040080.
目的: 获取适用于广西莪术简单序列重复(SSR)分析的高质量DNA
为该品种的遗传多样性研究提供参考。 方法: 选择广西莪术嫩叶为材料
采用经典十六烷基三甲基溴化铵(CTAB)法和改良CTAB法提取广西莪术总DNA
考察手工和机器研磨的差异性
采用UV检测DNA浓度和纯度
DNA作为模板进行聚合酶链反应(PCR)扩增
引物选择clest SSR-01
clest SSR-03
clest SSR-06
clest SSR-08
clest SSR-14
clest SSR-15
利用聚丙烯酰胺凝胶电泳检测DNA扩增效果。 结果: 改良CTAB法所提DNA的A260 nm/A280 nm 1.8~2.0
蛋白质、多糖、RNA等物质去除较彻底
DNA符合PCR扩增结果要求
使用姜黄属通用引物能扩增出清晰条带。经典CTAB法所提DNA的A260 nm/A280 nm 1.50~1.60
含较多杂质
无法用于PCR扩增等分子生物学研究。 结论: 改良CTAB法提取所得DNA质量较好
可有效去除次生代谢产物对DNA的干扰
适用于广西莪术SSR分子标记和遗传多样性分析。
Objective: To obtain high quality DNA from Curcuma kwangsiensis for simple sequence repeat(SSR) analysis
and provide a reference for research of genetic diversity of this variety. Method: Taking C. kwangsiensis leaves as experimental material
total DNA was extracted by the classic cetyl trimethyl ammonium bromide(CTAB) method and the modified CTAB method
difference between machine grinding and hand grinding was investigated
concentration and purity were determined by UV.DNA as a template for polymerase chain reaction(PCR) amplification
taking clest SSR-01
clest SSR-03
clest SSR-06
clest SSR-08
clest SSR-14 and clest SSR-15 as primers
polyacrylamide gel electrophoresis was adopted to detect DNA amplification effect. Result: When DNA extracted by the modified CTAB method
A260 nm/A280 nm of them was 1.8-2.0
proteins
polysaccharides
RNA and other substances were removed completely
DNA conformed to requirements of PCR amplification result
it could amplify out clear bands by C. universal primers and was suitable for SSR analysis.A260 nm/A280 nm of DNA extracted by the classic CTAB method was 1.5-1.6
it contained some impurities
which cloud not be used for PCR amplification and other molecular biology researches. Conclusion: DNA quality is good which was extracted by the modified CTAB method
this method can effectively remove interferenceof the secondary metabolites for DNA
it is suitable for SSR molecular marker and genetic diversity analysis analysis of C. kwangsiensis.
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