Baculovirus expression problem - Mixture of Bacmid DNA (Aug/13/2008 )

hi,

I am working on baculovirus expression system to express a human protein. My problem is after I transform gene construct into DH10Bac Ecoli. I got several white colonies, after I restreak them, they are still while. So I purified the bacmid DNA and confirmed with PCR by M13 primer, unfortunately, I got the bands of both recombinant bacmid and empty bacmid. I transfected the gene into sf9 cell, and after 48 hrs I checked both the cell and medium with westernblot, no protein expression. I think what I could do is either redo transformation and get pure recombinant bacmid and do transfection again or infect sf9 cell with the virus stock I have from transfection and try to amplify the virus stock and check protein expression again with high tire of virus. As I am new in insect cell expression field, could anyone tell me what I should do next? Thanks!!!

"I got the bands of both recombinant bacmid and empty bacmid."

This is strange. Using the plasmid you have, do transformation again and pick the correct colony. When picking colony for culturing in broth, just scractch a bit, try not to get contact with the agar too much.

While waiting for the result, try to check for your recombinant transfer plasmid construction if there is any problem.