Just a quick question - over the last couple of months, virtually none of our transfections with transfer plasmid DNA and Flashbac linearised DNA into Sf9 cells have yielded high-titer virus. Bacmids from the Bac-to-Bac system don't suffer this fate. The inability is independent of operator and construct. The P1 titer is around 10(3)-(4) pfu/ml (which is virtually background in qPCR) and don't amplify further from that.
We have bought in fresh linear DNA for OET, but to no avail. Our back-log is growing, users becoming impatient...and you know the rest...
Last time we tried it, I noted that the "transfected" cells were swollen and easily detached from the 6-well plates after 5 days of incubation as you normally only see when the cells are infected with a high-dosage of virus. Could there be some toxicity associated with the Flashbac DNA ?
I just wonder whether anyone else out there has experienced similar issues - we had a similar spell last summer, which then disappeared again without us pin-pointing the problem.
Thanks and hope to see many of you in Basel,