Change the way you think about Cloning….Discover In-Fusion®
Tuesday 8 September 12:30 to 13:15 - Hall 10B
Dr. Malathi Raman, European Cloning Product Manager, Takara Clontech, France
Dr. Louise Bird, Senior Research Scientist, Oxford Protein Production Facility (OPPF), University of Oxford, UK
Cloning is essential to most experiments carried out in molecular biology laboratories today. However, it is not usually the main focus but is rather an essential first step on the way to running the actual experiment and meeting specific research goals. A fast and accurate cloning method would thus be advantageous to minimize the time and resources spent on this initial cloning stage. The majority of researchers continue to use traditional ligation based cloning which can be time consuming and cumbersome due to several inherent limitations. These include the availability of specific restriction sites, the need to screen a considerable number of colonies before obtaining positive clones with the insert in the correct orientation, and the potential need for sub-cloning, since it is not always possible to directly clone into the final destination vector. However, Takara Clontech provides In-Fusion® HD Cloning Plus, a faster and more highly efficient alternative cloning technology to traditional ligation based cloning. In-Fusion® enables fast, directional, sequence independent and seamless cloning of any PCR fragment—or multiple fragments—into any linearized vector at any locus in a single 15 minute reaction. Researchers are not limited by restriction site availability as the In-Fusion® HD Enzyme fuses PCR-generated insert sequences and linearized vectors efficiently and precisely, utilizing a 15 bp overlap at their ends. This 15 bp overlap can be engineered by designing custom primers for the amplification of the desired sequences. The high >95% efficiency of In-Fusion® HD across a wide range of insert sizes (20 bp oligos to 15 kb fragments), also results in far fewer colonies needing to be screened before obtaining positive clones. Moreover, this cloning is seamless as no extra restriction site derived bases are added to final constructs, a crucial advantage when generating mutant or protein expression constructs as the addition of unwanted bases/amino acids is avoided. Sub-cloning is also unnecessary, since In-Fusion® always allows the direct cloning of single or multiple fragments into the final destination vector. Finally, In-Fusion® is also extremely versatile as it has been successfully used in many different applications including multiple fragment cloning, site directed mutagenesis (insertions, deletions and substitutions) and high throughput cloning for protein expression and purification studies.