Adeno-X Adenoviral System 3 - Simple Protocol
Constructing recombinant adenovirus with In-Fusion technology. DNA sequences can be rapidly transferred as PCR products to any pAdenoX vector using the In-Fusion cloning method. In this example, your gene of interest is amplified with 15 bp extensions that are homologous to the ends of the linearized adenoviral vector. The PCR product is then purified and mixed with the linearized adenoviral vector of choice in the In-Fusion reaction. Following the reaction, a portion of the mixture is transformed into E. coli (Stellar Competent Cells) and screened. Once a PCR-positive clone is identified, the recombinant pAdenoX vector is amplified, purified, and subsequently linearized with the restriction enzyme PacI, then transfected into Adeno-X 293 cells for viral rescue and amplification. Adeno-X GoStix can be used to determine the status of adenovirus rescue.