کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2058688 | 1543969 | 2014 | 7 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Recombination-assisted megaprimer (RAM) cloning Recombination-assisted megaprimer (RAM) cloning](/preview/png/2058688.png)
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 1 M betaine to enhance both reaction specificity and yield.
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Journal: MethodsX - Volume 1, 2014, Pages 23–29