Mathieu, JacquesAlvarez, EmiliaAlvarez, Pedro J.J.2014-08-012014-08-012014Mathieu, Jacques, Alvarez, Emilia and Alvarez, Pedro J.J.. "Recombination-assisted megaprimer (RAM) cloning." <i>MethodsX,</i> 1, (2014) Elsevier: http://dx.doi.org/10.1016/j.mex.2014.05.001.https://hdl.handle.net/1911/76328No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-joining. This new method, recombination-assisted megaprimer (RAM) cloning, significantly extends the application of restriction-free cloning, and allows efficient vector construction with much less time and effort when restriction-free cloning fails to provide satisfactory results. The following modifications were made to the protocol: 1) Limited number of PCR cycles for both megaprimer synthesis and the cloning reaction to reduce error propagation; 2) Elimination of phosphorylation and ligation steps previously reported for cloning methods that used exponential amplification, through the inclusion of a reverse primer in the cloning reaction with a 20 base pair region of homology to the forward primer; 3) The inclusion of 1 M betaine to enhance both reaction specificity and yield.engThis is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).Journal articlemegaprimerrestriction-free cloningPCR cloningexponential amplificationhomologous end- joininghttp://dx.doi.org/10.1016/j.mex.2014.05.001