How does In-Fusion Cloning work?
How does In-Fusion Cloning work?
| In‑Fusion Cloning requires 15 bp of overlap at the termini of the cloning insert and linearized cloning vector, or adjacent cloning inserts if multiple inserts are to be joined simultaneously. For multiple-insert cloning, we recommend increasing the overlap to 20 bp. These 15-bp homologous overlaps can be generated by PCR amplification or oligo synthesis of either of the cloning components. Homologous overlaps shorter than 12 nt or longer than 21 nt are not recommended. Translational reading frame continuity of a fusion construct can be adjusted by adding nucleotides between the insert-specific sequence and 15-nt overlap. 15-bp complementary regions must be located at the termini of adjacent DNA fragments or they will not be joined by In‑Fusion Cloning. The In‑Fusion enzyme mix generates single-stranded 5' overhangs at the termini of the cloning insert and linearized cloning vector. These overhangs are annealed at the sites of complementarity, and the recombinant circular construct is rescued in E. coli. (We do not recommend use of cells with competency less than 108 cfu/µg supercoiled DNA.) In‑Fusion Cloning does not allow for the covalent assembly of linear DNA molecules. |