Several companies have introduced recombination-based vectors to expedite cloning. While these systems do allow rapid cloning, they depend on specific sequences within the target vectors and, thus, restrict the user to a particular series of vectors. Clontech’s In-Fusion PCR Cloning Kit is a flexible system designed to be compatible with any desired vector. PCR fragments are simply incubated with the linearized vector and the BD Dry-Down In-Fusion PCR Cloning Reaction Mix. The reaction is then transformed into competent E. coli cells.
To test the effectiveness of this kit in our lab, PCR primers were designed to accommodate the 5' overhang of the Xho I linearized vector (as outlined in the manufacturer’s protocol). The cDNA of interest was amplified using EMD Bioscience’s KOD Polymerase. The vector was linearized by digesting with XhoI (NEB) and 5' phosphates were removed with Calf Intestinal Phosphatase (NEB). The PCR product and dephosphorylated linearized vector were isolated using Perfectprep Gel Cleanup Kit (Eppendorf). One aliquot of the BD Dry-Down In-Fusion PCR cloning reaction mix was reconstituted with 8.5 ul H2O, 1 ul vector (300ng) and 0.5 ul PCR product (100ng). The reaction was incubated for 30 min at room temperature and then 0.5ul of the In-Fusion reaction was transformed into XL-1 Blue Supercompetent Cells (Stratagene). The entire transformation was plated onto LB-ampicillin plates and incubated at 37C overnight. The reaction yielded approximately 100 colonies. Colonies were picked and plasmid DNA was isolated using FastPlasmid Mini (Eppendorf). One hundred percent of the colonies were positive for the insertion of the PCR product.
It took several failed attempts before the kit actually worked for us. On the first attempt, the primers were not designed quite correctly. Even after correcting this mistake, the second attempt failed, as we later found that one of the primers had a base that was different from the original order. Once again, we re-ordered the primers and found that we still did not obtain colonies after transformation using lab prepped electrocompetent cells. Ultimately, the In-Fusion reaction with the correct primers was transformed into XL-1 Blue Supercompetent cells from Stratagene and was successful.
Kerri A. Mowen, Ph.D.
Assistant Professor
Department of Immunology
The Scripps Research Institute