Hi all, I have a peculiar problem. I'm trying to select for a plasmid that: A) Confers Kanamycin resistance B) Bears a fusion protein containing wildtype GFP
I made up Kanamycin plates with 50ug/ml kan, which had recently been made & filter-sterilised from kanamycin sulphate powder. The powder is, I believe, about a year old, and has been stored in the fridge according to packaging instructions. The stock solution (50ug/ml) was stored at -20C once filtered into eppies. The cultures were DH10B, isolated from a Top10 kit, which had been left in LB in a fridge since February/March. I first broke them out into fresh broth, then subcultured for transformation to get exponential-phase cells. I expected a pretty idiot-proof transformation (with PEG-3350 & MgSO4) of E.coli DH10B, followed by selection of fluorescent green, kanamycin-resistance cells. The transformation procedure is as per: https://github.com/cathalgarvey/biohacking-protocols Instead, I got growth on transformant-plated plates *and* on negative control plates, which were treated identically but with only added T.E. rather than DNA solution. Growth is still as single colonies after spreading, rather than a lawn, but is pretty equally abundant on both plates, indicating some background resistance to whatever concentration of Kanamycin I'm using. Weirder still, when lit by blue light and filtered with an orange filter, nothing distinguishes the cells.. but when illuminated with a cheap handheld UVA torch, many of the colonies on *both* plates are bright fluorescent orange. The intensity of the orange appears to increase with intermittant exposure to UV. To ascertain whether the cells are expressing some orange pigment only upon UV-induced quorum sensing (as it's very clearly a colony-specific trait), I streaked an orange colony out beside a non-orange colony (again on kanamycin TB plates), and the results indicated some genetic factor: the orange colony lead to orange colonies, and the non-orange colony lead to almost exclusively non-orange colonies, bar one.. which might just be contamination from the other side. So, I'm baffled. On the one hand, why is my kanamycin so terribly non-selective? Any thoughts on powdered kanamycin stability? On the other hand, what are these fluorescent orange cells? They are identical to normal E.coli colonies to the naked eye, barring this vibrant orange fluorescence. I'm not even going to ask why my plasmid might be failing to transform/select. It would seem I have bigger problems. Thanks, Cathal -- www.indiebiotech.com twitter.com/onetruecathal _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
