yesterday's BD complaint and other things

[asimplechord]

But BD is on my hit list right now.

We're trying to do this assay for physiological relevance of the signaling proteins we study. It requires a fluorescent label in cells that are positive for the gene (or knockdown of the gene) involved. Now, I've got GFP-fused constructs of these genes, but they don't localize appropriately (in comparison with endogenous protein or myc- or HA-tagged protein), and at least one of the gene products' functions is adversely affected by the GFP moiety. So we went looking for a vector that had GFP with a stop codon and then an internal ribosome binding site (an IRES plasmid) so that GFP would be transfected with but not attached to our gene, making the cells positive for fluorescent staining without altering subcellular localization or function.

BD Bioscience produces this plasmid. It costs ~$500 for a few micrograms. Expensive, but what can you do? It would cost way more than that for me in terms of time and supplies to reengineer a vector we already have to do the same job. So we bought it. Subcloned four different genes into the second IRES site. Sequenced. Transfected, fixed, looked for green under the 'scope. No green cells.

Hmph. OK, did we do something wrong with the transfection? The fixing? Repeat. Still no green cells.

Called BD.

Tech services' response: It won't look green until you've subcloned something into the vector.

Us: Um, okay, done that. Still not green.

BD: Oh, you just didn't transfect well enough.

Us: Don't think that's the problem: our positive control for transfection efficiency (GFP in a different vector background) worked. But we have no GFP in the IRES samples. As in, zero positive cells.

BD: Well, this vector doesn't work for all gene products.

Us: We've tried four. None of them worked.

BD: Well, it's well known that secondary IRESes don't always work. Too bad.

Essentially, they gave us the technical services' equivalent of Fuck off. We've got your money, you've got our plasmid; it's not our fault if it doesn't work on a fairly regular basis. Screw you.

So. Out the money, out the time spent making the constructs and testing them. And there aren't really any other versions available for purchase. Bastards.

Other babble:

- Was anybody else weirded out by the Jane Fonda thing on TCR the other night? (I fell asleep on the sofa when it was originally aired and didn't actually see it until last night's rebroadcast, so I'm a day behind.) That was just... not right. It was completely not as cool as the original visit that she and Gloria Steinem made to the show. I felt uncomfortable watching it. Uncomfortable for him and embarrassed for her.

- I've got more ideas than motivation right now. (*cough* Tabby *cough*) And I definitely don't need to go check out the re-opened claims at slashfest.

Comments

[silentauror.livejournal.com]

Lol. She has, indeed. He really did look uncomfortable, though. It was just too much. And then the toss the next day was amusing, but still - when Jon asked how Stephen's lap was doing that day and Stephen replied, "empty", I have to admit that my gutter-ridden mind went straight to the sexual connotation and my eyebrows shot up in a most unimpressed manner - reminiscent of McGonagall, even. Then I realized that he just meant empty as in, devoid of Jane Fonda, not, erm, the other. *cough* Um. Still unimpressed, though. Lol.