Skowronski E W, Armstrong N, Andersen G, Macht M, McCready P M
Lawrence Livermore National Laboratories, CA, USA.
Biotechniques. 2000 Oct;29(4):786-8, 790, 792. doi: 10.2144/00294st05.
We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.
我们开发了一种快速的微孔板形式的质粒分离程序，用于为高通量DNA测序操作纯化测序级DNA模板。采用改良的溶菌酶/煮沸法制备含质粒的上清液，然后通过铁珠捕获进行纯化。结合后，珠子在磁场中沉淀，洗涤，DNA用水洗脱。该方法从深孔微孔板中1 mL过夜培养物中可产生高达10微克的质粒DNA。该程序适用于大规模实验，易于自动化，不需要昂贵的试剂或设备。整个方案可在短短2小时内完成，一名配备96孔移液器的技术人员每天最多可处理48个板。该方案对于任何模板数量、质量和可重复性至关重要的高通量操作来说都是理想的。
