How do I design PCR primers carrying 15-nt overhangs complementary to the termini of the linearized vector or adjacent insert?
How do I design PCR primers carrying 15-nt overhangs complementary to the termini of the linearized vector or adjacent insert?
Each forward (5' ? 3' sense strand) and reverse (5' ? 3' antisense strand) PCR primer should include the following:
-A 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). For multiple-insert cloning, we recommend increasing the homology to 20 nt. 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.
-(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.