Gateway LR reaction

Hello all,

Has anyone here every noticed something weird when doing a LR reaction in terms of getting some sort of mixture of plasmids? Like some sort of hybrid plasmid or multiple plasmids in 1 "cel" ?

I get a very low efficiency after the LR reaction (only a few cells per plate). Which is already a sign something is off.

The destination vector/donor vector are fine, but after the LR reaction I get only a few colonies and when I sequence them I get something really strange.
I use 3 primers:
1 primer on my destination vector before the GOI (primer 1) going through this GOI, the attb2 site and further on.
1 primer after my GOI on the destination vector going in the opposite direction (primer 2).
1 primer on the GOI , going towards my destination vector , going to the attb1 site (primer 3).

The first 2 primers give me what I want:
Primer 1: piece of my destination (now expression vector) , attb1 site, followed by the GOI and the attb2 site and my vector.

Primer 2: piece of my destination vector , attb2 site, followed by the GOI and the attb1 site.

The above primers, "added" together give me: destination vector - attb1 - GOI - attb2 - destination vector.
So far so good.

However: using primer 3 (that binds in my GOI) I get : GOI followed by a little piece of the attb1 site and then the donor vector...
This makes no sense to me.

Do I have some sort of hybrid plasmid? But how is this possible? Or do I have a mixture of plasmids? But this seems also weird since its from a single colony (I hardly have any colonies on my plate anyway so its really form a single CFU).


I know that something is wrong since I only get a few colonies per LR reaction after plating, but I really do not get this hybrid/weird plasmid.

A note: when checking the quality of the sequenced results from the primer that binds my GOI (primer 3) I do see that the peaks are not good when I reach the attb1 site (when it goes from GOI to the donor vector rather than expression vector).

So I am guessing I have 2 plasmids rather than 1.

Anyone an insight in this?