Related Experiment Video
Updated: Apr 7, 2026
09:02
Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
Published on: January 8, 2015
17.2K
Recombination-assisted megaprimer (RAM) cloning
Jacques Mathieu1, Emilia Alvarez1, Pedro J J Alvarez1
1Department of Civil and Environmental Engineering, Rice University, Houston, TX, United States.
Methodsx
|July 8, 2015
Summary
Researchers developed recombination-assisted megaprimer (RAM) cloning, a faster and more reliable molecular cloning method. This technique improves upon restriction-free cloning, offering enhanced efficiency and reduced effort for vector construction.
Area of Science:
- Molecular Biology
- Biotechnology
Background:
- Molecular cloning is essential for genetic engineering but existing methods have limitations.
- Restriction-free cloning is efficient but lacks universal reliability.
- Complex, laborious, and expensive cloning techniques hinder research progress.
Purpose of the Study:
- To enhance the reliability and efficiency of restriction-free cloning.
- To develop a novel molecular cloning method with improved success rates.
- To streamline vector construction for broader research applications.
Main Methods:
- Modification of restriction-free cloning using exponential amplification and homologous end-joining.
- Development of recombination-assisted megaprimer (RAM) cloning.
- Optimization of PCR cycles, primer design, and reaction conditions including the addition of 1 M betaine.
Main Results:
- RAM cloning significantly improves the success rate of restriction-free cloning.
- The method reduces the time and effort required for efficient vector construction.
- Elimination of phosphorylation and ligation steps simplifies the protocol.
Conclusions:
- RAM cloning offers a more robust and efficient alternative when standard restriction-free cloning fails.
- This technique broadens the applicability of restriction-free cloning for molecular biology research.
- The optimized protocol minimizes errors and enhances reaction specificity and yield.
Related Concept Videos
02:53Conservative Site-specific Recombination and Phase Variation
7.4K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
7.4K
02:31Homologous Recombination
65.7K
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
65.7K