I have been having difficulties with low transduction efficiencies of my retrovirus production. I expand my plasmid of interest (on MiG-GFP plasmid) in DH5α E Coli for ~24 hours, purify with Qiagen midi prep kits, then put on ~90% confluent 293FT cells with either retronectin or polybrene for 24 hours. I then change media and harvest supernatant at 48 and 72 hours of incubation with the 293FT cells. I then expose 3T3 cells to varying volumes of the virus containing 293FT media and am able to transduce about 10% of the 3T3 cells, which is really low. That being said, here and there I am able to get very high transduction efficiencies on the 3T3 cells, but for unknown reasons. I have tried concentrating the virus but transduction efficiencies don't increase all that much. And I have tried growing up the plasmid in Stbl3 cells, which if anything gives me worse virus.
Anything here jump out as being responsible for low transduction efficiencies. Or is there another way somebody does this that works really well?