CTAB法提取质粒DNA的探讨

Discussion CTAB method to extraction of plasmid DNA

目的利用CTAB性质,建立一种快速、简单、高效的质粒DNA提取方法。方法从CTAB的浓度、时间、pH值、温度和盐溶液浓度几个方面进行质粒DNA提取方法的探讨,与传统碱裂解方法进行比较,经DNA条带回收,PCR实验扩增已知插入片断,来验证CTAB法提取细菌质粒DNA的实验效果。结果 CTAB法提取质粒DNA,在质和量方面均优于碱裂解法。单因素法对CTAB提取质粒方法探讨,最适CTAB浓度、pH值、水浴时间、温度分别为0.6%(w/v)、7.0,15 min,60℃。虽然4×CTAB也有较好效果,但需加1.5 mol/L的Na Cl溶液,才利于上清液的分离。PCR结果进一步验证DNA的纯度和性质。结论 CTAB法提取质粒DNA方法简单、快速,质量和纯度完全可以替代传统方法用于分子生物学后续实验。

Purpose： To establish a rapid, simple and efficient method for the extraction of plasmid DNA by using the CTAB property. Methods ：The study was made by the method of extracting plasmid DNA from CTAB concentration, time, pH value, temperature and concentration of salt solution, and compared with the traditional alkaline analysis method. DNA band recovery and PCR experimental amplification of known insert fragments were used to verify the experimental results of CTAB method to extract bacterial plasmid DNA. Results ： CTAB extraction of plasmid DNA were better than alkaline analysis method in terms of quality and quantity. By the single factor method for extraction of plasmid CTAB method, the optimum CTAB concentration, pH value, time and temperature of water bath were 0.6% （w/v）, 7, 15min and 60℃. Although 4 × CTAB also had a good effect, but NaC1 1.5mol/L solution need be added, and is conducive to the separation of the supematant. PCR resuits further verified the purity and properties of DNA. Conclusion： CTAB method is simple and rapid, and the quality and purlty of plasmid DNA can be replaced by traditional methods for molecular biology experiment.