Good day. I am using the Clontech Adeno-X system to generate recombinant adenoviruses to use as vaccines, but I have experienced a few technical issues along the way. Currently I need to digest the recombinant vector with PacI to expose the ITR regions for virus replication after transfection but the PacI digest is either not yielding a complete digest or cutting in the wrong place as the cut-out DNA fragment is much larger than expected. Has anyone else had this problem?

I have tried transfecting with the recombinant virus backbone and I get good transfection efficiency but as soon as I harvest after CPE I do not get any infection- thus I assume no virus being produced. When I do the PacI digest I am supposed to see a 3,5kb fragment that was cut out and the rest of the 35kb backbone i the wells of the agarose gel. I however see fluorescence in the wells and a 21kb fragment on the gel. Suppliers of backbone say it is incomplete digest of plasmid? This does not make sense to me. How did you fix this?