摘要
碱法制备的低拷贝质粒因含有大量杂质而无法获得有效的测序结果。为此,以纯λDNA为样品,对硅藻土纯化DNA的方法进行了优化,表明硅藻土悬液的最佳用量是20μl/μgλDNA,回收率达77.31%。应用此改良硅藻土法纯化一长为14953bp的低拷贝质粒pLZ14,所得质粒纯度达23.87%,比未纯化前提高了11.06倍。经纯化的pLZ14质粒测序信号强、无误认碱基,而未经纯化的质粒测序时信号弱、错误普遍存在。
Sequencing of low copy number plasmid DNA failed quite often due to high amount of impurities in sample. To develop an efficient and highly repeatable DNA purification protocol especially suitable for low copy number plasmid DNA, the amount of diatomaceous earth suspension applied to bind DNA was optimized. It was found that 1 μg of pure λDNA was most efficiently recovered when 20 μl of the suspension was applied, with a recovery as high as 77.31%. pLZ14, a low copy number plasmid of 14 953 bp, was purified 12.06-fold with the improved protocol, reaching a purity of 23.87%. Strong, clear sequencing signals and correct results were obtained with the purified pLZ14 plasmid DNA, while weak, confusing signals and universal errors were found for DNA from routine alkaline lysis and purification protocols.
出处
《细胞生物学杂志》
CSCD
2005年第6期679-683,共5页
Chinese Journal of Cell Biology
基金
国家自然科学基金(No.30370989
No.30100125)
浙江省自然科学基金(No.301291)资助~~
关键词
硅藻土
盐酸胍
质粒
DNA纯化
diatomaceous earth
guanidine hydrochloride
plasmid
DNA purification
