What are PCR primer design considerations for compatible with In-Fusion Cloning?
What are PCR primer design considerations for compatible with In-Fusion Cloning?
Each forward (5' - 3' sense strand) and reverse (5' - 3' antisense strand) In?Fusion Cloning PCR primer should include the following:
-Template-specific (gene-specific) portion at its 3' end. To ensure specific and efficient PCR amplification, the template-specific portion of the primer should be 18–25 nt in length.
-15 nt of homology at the 5' end of the primer, complementary to the termini of the linearized vector or adjacent inserts (if multiple inserts are to be cloned simultaneously). Homologous overlaps shorter than 12 nt and longer than 21 nt are not recommended. The 15-bp complementary regions must be located at the termini of adjacent DNA fragments or they will not be joined by In?Fusion Cloning.
*We recommend increasing homology to 20 bp for multiple-insert cloning.
*When a vector has been linearized via restriction digest, the 5' overhang of a restriction site is included in the 15 nt of homology, while the 3' overhang of a restriction site is excluded from the count of the 15 nt of homology.
-(Optional) To ensure continuity of the translational reading frame, or to preserve restriction site(s), additional nucleotides can be added to the PCR primer(s) between the template-specific portion and the 15-nt homologous overlap.