Recombination-assisted megaprimer (RAM) cloning

Jacques Mathieu,Emilia Alvarez,Pedro J.J Alvarez

doi:10.1016/j.mex.2014.05.001

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:•Limited number of PCR cycles for both megaprimer synthesis and the cloning reaction to reduce error propagation.•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.•The inclusion of 1M betaine to enhance both reaction specificity and yield.

Highlights

Since the discovery of restriction enzyme-based methods for plasmid construction [1,2], researchers have sought to improve upon the efficiency, reliability, and cost of molecular cloning
(1) If RAM cloning is being performed subsequent to an attempt to perform RF-cloning, the same megaprimer can be used that was produced for the original RF-cloning, and this step can be bypassed
While RF cloning may be less expensive per reaction based on reagent costs, we found that in many cases it would have been more economical to utilize RAM cloning over RF cloning due to the increased amount of screening we have had to perform for some cloning reactions

Summary

Method details

RAM cloning is designed to work as either a stand-alone method, or as a means to salvage a failed restriction-free (RF)-cloning attempt. Use the same tip to streak a 1% agar plate containing the appropriate antibiotic Perform this step for the desired number of colonies and incubate the plate overnight at 37 8C. (3) PCR amplify the desired insert region using the following conditions: an initial denaturation step (35 s, 98 8C), followed by 30 cycles consisting of a denaturation step (35 s, 95 8C), an annealing step (35 s, average primer Tm), and an extension step (30 s/kbp, 72 8C). Follow this with a final extension step at 72 8C for 5 min. While RF cloning may be less expensive per reaction based on reagent costs, we found that in many cases it would have been more economical to utilize RAM cloning over RF cloning due to the increased amount of screening we have had to perform for some cloning reactions

Background

Findings

Methods