How should I plan my In-Fusion experiment
How should I plan my In-Fusion experiment
Successful In-Fusion Cloning reactions require 15-bp homologous overlaps at the termini of the cloning insert and linearized vector, or adjacent inserts if multiple inserts are to be joined simultaneously. We recommend increasing the overlap to 20 bp of homology for multiple-insert cloning.
-These overlaps can be generated by PCR amplification or oligo synthesis of either of the cloning fragments.
-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 homologous overlap.
-Complementary regions must be located at the termini of adjacent DNA fragments or they will not be joined by In-Fusion Cloning.
A 3' exonuclease in the In-Fusion enzyme mix generates single-stranded 5' overhangs at the termini of the cloning insert and linearized vector. These overhangs are annealed at the sites of complementarity, and the recombinant circular construct is rescued in E. coli.
-We do not recommend the 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.