Rapid Resuspension of Pelleted Bacterial Cells for Miniprep Plasmid DNA Isolation

Small-scale purification of plasmid DNA is commonly used in molecular biology procedures. Several protocols for rapid isolation of DNA have previously been published (1,2). Several companies have also developed reliable miniprep kits that have become the method of choice for most laboratories when preparing plasmid DNA. However, even when using a kit, these miniprep processes can be laborious and time-consuming, particularly when large numbers of minipreps are performed in parallel. For multiple plasmid preparations, the rate-limiting step of miniprep protocols is resuspension of the cell pellet. The standard technique is to pellet multiple bacterial cell cultures in 1.5-mL microcentrifuge tubes for 30 s to 10 min, depending on the volume of culture pelleted. After centrifugation, the cells are resuspended by vortex mixing. Complete resuspension of cells before lysis is critical to achieve a good yield. However, this process often requires 20 s to several minutes to achieve complete resuspension. We have devised a rapid protocol to completely resuspend the cell pellet in only 3–4 s without any negative effect on the quantity and quality of plasmid DNA, irrespective of size. E. coli cells carrying plasmids of 3.0, 5.6 or 20 kb in size (pBluescript [Stratagene, La Jolla, CA, USA] plus Time of Cell Volume Scraping Centrifugation of Culture Vortex Rack