Brun Y V, Breton R, Lapointe J
Départment de biochimie, Faculté des sciences et de génie, Université Laval, Québec, Canada.
DNA Seq. 1991;1(5):285-9. doi: 10.3109/10425179109020784.
We have developed a simple rapid plasmid DNA mini-preparation method which yields DNA of sufficient quality to be used in large scale sequencing projects. The method, which is a modification of the alkaline method of Birnboim and Doly (1979), requires less than two hours. We have eliminated the use of organic extractions, RNase digestion and alkaline denaturation of the DNA for annealing of the primer. The proportion of supercoiled plasmid DNA obtained is close to 100%. Greater than 80% of the clones yield at least 500 bp of sequence information per primer. The sequencing reactions from these double-stranded templates can be done on both strands using the universal and reverse sequence primers with the usual two reactions per primer, one to read close to the primer and one to read far from it. Thus, each clone yields at least 1 kb of sequence information. The preparation of the templates and the sequencing reactions can be done in less than three hours so that the sequencing gel can be run the same day.
我们开发了一种简单快速的质粒DNA小量制备方法，该方法所产生的DNA质量足以用于大规模测序项目。此方法是对Birnboim和Doly（1979年）的碱性方法的改进，耗时不到两小时。我们省去了有机抽提、RNA酶消化以及用于引物退火的DNA碱性变性步骤。所获得的超螺旋质粒DNA比例接近100%。超过80%的克隆每个引物至少能产生500 bp的序列信息。使用通用引物和反向序列引物，从这些双链模板进行测序反应时，每条引物通常进行两个反应，一个用于读取靠近引物的序列，另一个用于读取远离引物的序列。因此，每个克隆至少能产生1 kb的序列信息。模板制备和测序反应可在不到三小时内完成，这样测序胶可在当天进行电泳。
