Cloning Woes: Dealing with Viral ITRs - (Dec/12/2014 )
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...
If you are doing colony PCR (which it sounds as if you are), then DNA from your ligation reaction can be present on the plate without being present in the cell. I'd suggest that this is what is happening. Likely this means you are ligating too high a concentration of vector and insert, and using too much of the ligation reaction in the transformtaion. Inevitably you will have very high background in this reaction with intramolecular ligation being very preferred over bimolecular ligations. I'd suggest re-plating your transformation plate prior to colony pcr (a replica plate might be easy). Better would be to redesign your cloning strategy to use two enzymes, or to go back and optimize your phophatase reaction. You should use a phosphatase that can be heat killed (SAP or antarctic phosphatase). Use the least amount possible (you may want to try a titration to dramatically reduce but not eliminate religation of your vector).