Problems with High Background with Gateway Cloning - (Jun/12/2010 )

Hello, I've recently started using Invitrogen's Gateway cloning system and I have been having some problems with high background in my -ve control LR recombinase rxns. In principle i should be getting NO background at all but I have been getting almost as many colonies in my -ve's as my +ve's. I have been transforming my rxns into TOP10 cells and as such any dest vectors which have not recombined should not persist as the ccdB gene should kill these bacteria. Also my entry vector is a different antibiotic resistance (Spec rather than Amp) than my dest vector so any bacteria containing these vectors shouldn't grow on AMP plates! It was suggested to me to try and make a new prep of my Dest vector in AMP+CAM plates/broth as the vectors apparently can kick out the GW cassette which could explain the background problem. However, having done this I see no improvement in the levels of background. Has anyone had similar problems and if so how did you overcome them? Thanks

PS. the problem seems to be specifically with the Dest vector as I used the same entry clones with other dest vectors with no problems. friends who have used this vector in the past tell me that high background was never a problem for them......

same problem. I checked all the possibilities I read in the forum and I had no results. I hope someone can help us.

Thank you in advance!!