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dc.contributor.authorMathieu, Jacques
Alvarez, Emilia
Alvarez, Pedro J.J.
dc.date.accessioned 2014-08-01T19:09:26Z
dc.date.available 2014-08-01T19:09:26Z
dc.date.issued 2014
dc.identifier.citation Mathieu, Jacques, Alvarez, Emilia and Alvarez, Pedro J.J.. "Recombination-assisted megaprimer (RAM) cloning." MethodsX, 1, (2014) Elsevier: http://dx.doi.org/10.1016/j.mex.2014.05.001.
dc.identifier.urihttps://hdl.handle.net/1911/76328
dc.description.abstract No 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.
dc.language.iso eng
dc.publisher Elsevier
dc.rightsThis is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
dc.rights.urihttp://creativecommons.org/licenses/by/3.0/
dc.title Recombination-assisted megaprimer (RAM) cloning
dc.type Journal article
dc.contributor.funder SENS Research Foundation
dc.citation.journalTitle MethodsX
dc.subject.keywordmegaprimer
restriction-free cloning
PCR cloning
exponential amplification
homologous end- joining
dc.citation.volumeNumber 1
dc.type.dcmi Text
dc.identifier.doihttp://dx.doi.org/10.1016/j.mex.2014.05.001
dc.type.publication publisher version


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