What's In-Fusion's compatibility with site-directed mutagenesis?
What's In-Fusion's compatibility with site-directed mutagenesis?
In-Fusion Cloning allows single or multiple base changes, deletions, and insertions through the use of inverse PCR. Please note this application relies on high-fidelity PCR. It is essential to use a PCR polymerase with high proofreading activity, such as PrimeSTAR Max DNA Polymerase. This polymerase is highly robust and accurate, enabling amplification of up to 6 kb of human genomic DNA, 10 kb of E. coli genomic DNA, and 15 kb of lambda DNA, and exhibits minimal error rates on GC-rich templates.
-Each PCR primer directs DNA synthesis in the opposite orientation of the other on a circular vector template.
-The 3' ends of the forward and reverse PCR primers are 18–25 nt that are complementary to the template, ensuring efficient and specific amplification.
-Mutations are incorporated within the homologous 15-nt overlap located at the 5' ends of the forward and reverse PCR primers (this homologous overlap is required for the recircularization of the mutated vector).
*Single- or multiple-base changes, deletions, or insertions can be introduced in a single In-Fusion reaction.
*A larger deletion of any desirable length can also be introduced by positioning the 3' ends of the forward and reverse primers at the border sites of a deletion, with homologous overhangs carried by the 5' end of either of the primers.
-The resulting inverse PCR will generate a linear double-stranded vector with 5' and 3' ends complementary to each other, and carrying the 15-nt homologous overlap (this overlap will be joined through the In-Fusionreaction and recovery in E. coli, thus generating a mutated vector).
-Vectors amplified with inverse PCR must be treated with Cloning Enhancer to destroy the parental vector. Additional purification by preparative gel electrophoresis may be required to ensure isolation of the linearized vector from PCR byproducts and possible remnants of the parental circular vector.
For additional details, please see our Mutagenesis with In-Fusion Cloning tech note.