Development of rapid microwave-mediated and low-temperature bacterial transformations
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ORIGINAL ARTICLE
Development of rapid microwave-mediated and low-temperature bacterial transformations Valerie T. Tripp & Johnathan C. Maza & Douglas D. Young
Received: 19 December 2012 / Accepted: 5 May 2013 / Published online: 21 May 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract The introduction of exogenous DNA into Escherichia coli is a cornerstone of molecular biology. Herein, we investigate two new mechanisms for bacterial transformation involving either the use of microwave irradiation or a freeze–thaw protocol in liquid nitrogen. Ultimately, both methods afforded successful transfer of plasmid DNA into bacterial cells, with the freeze–thaw technique yielding efficiencies of ~105. More importantly, both techniques effectively eliminated the need for the preparation of competent cells. Keywords Microwave irradiation . Bacterial transformation . Molecular biology
Introduction The introduction of exogenous DNA into bacterial cells is a cornerstone of molecular biology and thus has become a fundamental technique in innumerable laboratories [1]. Bacterial transformation has been the subject of a variety of investigations as it is essential for applications involving molecular cloning, mutagenesis, protein expression, and many others [2]. Currently, there are several known methods for efficient introduction of the exogenous DNA into a variety of bacterial species; however, each has distinct advantages and disadvantages, requiring the necessity for further studies to improve this prevalent technique [1]. Transformation protocols generally involve a three-step
Electronic supplementary material The online version of this article (doi:10.1007/s12154-013-0095-4) contains supplementary material, which is available to authorized users. V. T. Tripp : J. C. Maza : D. D. Young (*) Department of Chemistry, The College of William & Mary, Williamsburg, VA 23187, USA e-mail: [email protected]
procedure: the preparation of competent cells, a physical shocking of the cells, and a recovery. The two most common methods for transformation of E. coli cells are chemotransformation and electroporation, each differing in their cell preparation and method of physical shock. We set forth to develop new methods of rapid transformation of bacterial cells using either low temperatures or microwave irradiation and shortening the protocol by the removal of a time-consuming competent cell preparation. Chemotransformation involves the pre-treatment of cells with multiple washings/incubations containing CaCl2 to aid in the uptake of plasmid DNA [3–5]. It is speculated that the divalent cation serves as a mechanism to shield the negatively charged DNA backbone from the charge of the phospholipid bilayer, increasing its cellular membrane permeability [6]. Typically, these chemically competent cells are then incubated on ice to solidify cellular membranes, exposing pores for DNA transport. In order to shock the cell to increase uptake, chemotransformations utilize a brief pulse of heat (often 42 °C for 45–60 s) followed by a recovery prio
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