Hi all,

Hope you are having a nice day! I was wondering if anyone has used the bac-to-bac baculovirus expression kit to express recombinant protein? There is a step when creating the virus, where you transform a shuttle vector into DH10bac competent cells which contain a bacmid. This recombines with the shuttle vector to introduce your gene of interest into the bacmid, which you then isolate. I have followed this procedure using the manufacturer's specifications, and do get colonies. When I screen my control (uses a control vector sent with kit), the recombination did occur, but did not occur with my gene. I picked six colonies from each. The control vector has an insert which is a similar size to my insert. You select with gentamicin (shuttle vector encodes resistance), tetracycline (helper plasmid which contains genes that help facilitate the recombination event), and kanamycin (bacmid has resistance gene). Blue/white selection with bluo-gal and IPTG is supposed to select for recombination, colonies with recombinant bacmid should be white, not blue. I bought new bluo gal for my plates, so definitely should be good, and am getting many colonies on my plate but none are blue (which again would be negative.) However, when I screen them, they contained unrecombined bacmid. I have just picked 20 colonies off of the plate and am screening with colony PCR. My next ideas if I don't get anything from that are to up the bluo gal, up the IPTG, and troubleshoot/ use more of my shuttle vector in case actual transformation is the problem (kit has me using 1 ng which to me, seems low for this application.) The cells are expensive however, and I don't know much about optimizing recombination/ratios, etc. Does anyone have any suggestions? Sorry for any incorrect terminology or statements, I am completely new to this system. Also, I have attached a schematic of the system from the manual in case this helps.

Thanks,

Claire

Also, sizes:

Shuttle vector + my gene: approx 8 kb

Bacmid: 135 kb