I'm attempting to propagate an Adeno Associated Virus, serotype 9 (AAV9) genome in a modified pFastBac1 (pFB1) vector (LifeTechnologies/Invitrogen). pFB1 is modified so that only the Tn7 recombination sites, Amp resistance, and replication origins remain; so it's more or less a traditional vector with Tn7 recombination sites. Restriction was done with only one enzyme, PacI, thereby allowing both orientations of insert (it is unimportant for the downstream purpose). No phosphatase was used (no yield when I attempted to phosphatase vector).
After transformation into Stbl2 cells, clones were grown overnight at 30C on LB+Amp 100ug/mL plates. Colonies were screened by PCR with primer "A" designed to anneal to vector and primer "B" designed to anneal to insert; therefore amplification is dependent on successful ligation (and a certain orientation of vector to insert).
I scanned 16 clones. Two produced an amplicon ~500bp. Based on my constructed maps, I was expecting an amplicon of ~550bp. However, because the amplicon was unique to two clones, I proceeded with a miniprep. Based on previous results (harvesting at OD600 = 2.2 A resulted in no plasmid yield), I allowed the culture (LB+Amp 100ug/mL, 30C 225rpm) to grow until OD600 = 4.0 A - representing a ~24 hour incubation (I diluted and measured A between 0.0-0.5, of course). I spun 1.5mL culture, and isolated plasmid. Restriction digest simply linearizes the plasmid, and the size suggests it is vector ligated on itself.
Anyone here cloned viral genomes before? What E coli strain do you use? Growing conditions (temp, rpm, media)? At what cell denisity/OD600 do you harvest?
Thanks for any advice - I've been at this for a month, and I'm at my wits end...