A Simple, Rapid Method for Extracting Large Plasmid DNA from Bacteria

摘要

We are studying the lateral transfer of transmissible antibiotic resistance plasmids among stream bacteria impacted by fecal runoff from poultry and cattle. Such plasmids are typically large (ca. 40 – 100 kb) and occur in low copy numbers in the cell and have therefore typically been difficult to isolate and therefore to study. Traditional protocols, based upon variations of the standard alkaline-lysis method, are long (ca. 1 1/2 to 2 days) and difficult. Commercial kits designed for the isolation of Baterial Artificial Chromosomes (BACs) can be used and are an improvement; however, these are expensive and still require hours of sustained effort. We have adapted a method published by Rondon et al. (1999), originally designed for the isolation of BAC DNA, for the rapid isolation of large plasmid DNA. In this method, lysis and alkaline denaturation steps are combined, incubation steps are vastly reduced, proteins are removed via a simple ammonium acetate/chloroform step, and the DNA precipitated using a plyethylene glycol/NaCl step. No ethanol precipitation is required. If additional purification is required, extracted DNA can be further processed through a Qiagen Plasmid Mini or Midi column (Qiagen Inc., Valencia CA). The method is rapid (under 1 hour), easy, very inexpensive and has been reliably used by undergraduate students to isolate large (up to 200 kb) native plasmids from a variety of both Gram-positive and Gram-negative genera including _Shigella_, _Klebsiella_, _E. coli_, _Pseudomonas_, _Bacills_, _Streptococcus_, _Staphylococcus_, and _Enterococcus_, as well as BACs from _E. coli_. The protocol is simple and reliable enough to be used for the rapid large-scale visualization of native plasmids and we have used it to visualize and isolate DNA from hundreds of multidrug resistance plasmids exogenously captured from stream sediments, soils, and beach sands.