A primer set homologous to the gene of interest and the vector is used to amplify a PCR product. This purified PCR product is incubated with restriction enzyme-digested vector and Reagent A of the PCR Cloning BioAssay™ Kit. Following a brief incubation with Reagent B, the annealed vector is ready for transformation.
Proper primer design is critical for successful PCR and transformation steps. Primers for the gene of interest should contain the restriction site as well as at least 10 nucleotides of sequence homologous to the vector sequence. Using two different restriction enzymes will allow directional cloning of the insert. Note: Once purified, the digested vector can be used with multiple inserts if the primers for insert amplification are designed correctly.
To make primers for PCR amplification of the gene or sequence of interest, at least 18 nucleotides of homology to the desired gene sequence is recommended.
Insertion of Multiple DNA Fragments: PCR, Cloning BioAssay™ Kit allows for the insertion of multiple DNA fragments into the desired vector using only a single reaction.
Adding or “Fixing” Mutations in DNA Fragments: The PCR, Cloning BioAssay™ Kit can also be designed to add or “fix” mutations within a gene of interest, using the protocol for multiple DNA inserts (above). We recommend the two PCR products share at least 18 nucleotides of homology.
Supplier Page from United States Biological for PCR Cloning BioGenomics™ Kit