| Autor: |
Jacques Mathieu, Emilia Alvarez, Pedro J.J. Alvarez |
| Jazyk: |
angličtina |
| Rok vydání: |
2014 |
| Předmět: |
|
| Zdroj: |
MethodsX, Vol 1, Iss C, Pp 23-29 (2014) |
| Druh dokumentu: |
article |
| ISSN: |
2215-0161 |
| DOI: |
10.1016/j.mex.2014.05.001 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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