七筋菇基因组DNA提取方法的比较研究  

Extraction of genomic DNA and optimization of template concentration for ISSR-PCR amplification of clintonia Udensis trautv.et mey

作　　者：王祎玲[1] 潘豪杰[1] 沈张波[1] 赵桂仿[1] 

机构地区：[1]西北大学生命科学学院

出　　处：《西北大学学报（自然科学版）》2007年第4期617-620,共4页Journal of Northwest University（Natural Science Edition）

基　　金：教育部博士点基金资助项目(20040697010)

摘　　要：目的对七筋菇基因组DNA提取和ISSR-PCR扩增模板浓度进行优化。方法以常规的CTAB法为基础,对七筋菇基因组DNA提取进行了优化:提取液中加入PVP(6%(w/v)),β-巯基乙醇的浓度提高至5%(w/v);第一次抽提后,重新加入提取液及抗坏血酸(20%(w/v)),65℃水浴30 min。结果所提样品DNA,A260/280,A260/230的平均光吸收值分别为1.78,1.99,浓度为105 ng/μL左右,ISSR扩增带多且清晰明亮,稳定性及多态性高,表明DNA纯度高;对扩增反应的最佳模板浓度进行研究表明,所提DNA模板稀释2倍效果最好(浓度为50 ng/μL)。结论改良后的CTAB法适于提取七筋菇植物的基因组DNA。Aim To optimize protocol for Clintonia udensis Trautv.et Mey.genomic DNA extraction and ISSR-PCR amplification template concentration.Methods An improved means,based on the general CTAB,was used to extract C.udensis Trautv.et Mey.genomic DNA：①PVP（6%（w/v））was added and β-mercaptoethanol concentration was 5%（w/v） in the solution;②Ascorbic acid（20%（w/v）） and extraction solution were added again after the first extraction,water bath for 30 min at 65℃.Results ①Templates from samples（1.78 of A260/280 ratio,1.99 of A260/230 ratio,105 ng/μL of concentration approximately） had more amplification bands which were clear,bright,stable and polymorphic,and it showed high purity of DNA.②The optimized concentration of templates used in ISSR-PCR amplification was diluted to two times（its concentration 50 ng/μL approximately）.Conclusion Genomic DNA of Clintonia udensis Trautv.et Mey.could extract by the improved CTAB method.

关 键 词：七筋菇 DNA提取 模板浓度 

分 类 号：Q949.718.23[生物学—植物学]