Gateway Recombination Problems - (May/24/2007 )

I have been trying to move my protein of interest from the Gateway entry clone (pcDNA 3.2/V5/GW/TOPO) into two different expression vectors (EmGFP and pDEST31) using the LR Recombination reaction. So far, the recombination reaction has been unsuccessful and I end up with just my entry clone in the end. Has anyone had this problem or have any suggestions?


We were also thinking that maybe linearizing or just using a fragment, rather than the super coiled entry clone might be more efficient - has anyone had experience with this?

Call Invitrogen Tech support...they should help!

check the cells that you use for transformation. The LR recombination sequence from invitrogen also has ccDB and after the reaction, you could transform in cells other than DB3.1 and all plasmids without ccDB will grow.

I let the LR reaction run overnight at room temperature. made a huge difference in getting successful recombinants
good luck!

Well, linearizing doesn't help...you've probably read that in the Gateway Cloning catalogue. Is your Clonase OK. You have to handle it precisely as they say. I have recently done this reaction...works beautifully. Also, once recombination is done, you do not use DB3.1 cells for transformation becoz your ccdB gene is gone if recombination has happened. Just use a lab strain of DH5alpha. You may not get a lot of colonies as the catalogue says. But the transformed ones are bigger than untransformed ones. Also you need an antibiotic in your LB plates.

Thanks for the replies:


scolix: we are using DH5alphas for recombination, the plasmids we purify out are usually the entry clone, so it doesn't recombine.

Bastila: Thanks for the advice, I will certainly try that with the next reaction.

Fledgeling: we're doing everything by the book, and only growing on Amp plates. The destination vector has a Bsd resistance gene so we are going to try double selection on the next experiment also. I will also take a look at our old plates and look for colony size, thanks!

Mous: Don't you think we have already called them? LOL

What antibiotic resistant are the entry clones? they should be different from the pcDNA , right ? else the recombination reaction & transformation might not work properly.

The entry vectors we have are kanamycin and the actual vector is ampicillin, so this should again not give you the entry clones.