Dotte

Well indeed. Its a very broad view and I find it weird they add them to the list if "interactions". Its a bit confusing and not very specific.

Ah yes, I see what you mean. It does mean that they have an effect on eachother, but it does not mean (necessary) that the proteins interact wich eachother (physically)?

But what does that mean?

Hallo all, I am checking a website (to check protein-protein interactions) and I have a problem understand what they mean with "genetic" vs "physical "interactions. See, for example, http://www.yeastgenome.org/locus/S000002706/interaction You can see a list with interaction and an interaction overview. But I have troubles understanding what they mean with "physical" and "genetic" interactions. The physical ones, I get: protein protein interactions in the cell as they are. But the "genetic" one, I am not 100% sure I get it. They explain it like this: (http://www.yeastgenome.org/help/function-help/interactions) Physical interactions are defined as the direct physical binding of two proteins, or co-existence in a stable complex, whereas genetic interactions are interactions between two or more mutants. For more details about BioGRID's curation process, see here. So what does this mean? A genetic interaction is an interaction proven after mutating one gene (protein) this first protein has another (stranger/other) effect after they changed a second gene as well. So by mutating a second gene they notice that the first one is also acting different compared to when its not mutated or mutated alone. Is this correct or ? Anyone that can elaborate on this? Thanks in advance

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?