Hi Cathal, I had a problem with Kan selection a year or so ago where I was seeing lots of pappilation (cells mutating to Kan resistance). At the time I was using 50 ug/ml which was/is the recommended dose. I tested several concentrations of Kan and a dose to 100 ug/ml in the plates seemed to solve the problem. Now when transforming I use 100ug/ml in the plates and that seems to provide a strong enough selection that I don't see any spontaneous Kan resistant colonies. Your DH10B stock seems pretty old, and you may have inadvertantly selected for cells that were resistant to low levels of Kan. I usually only keep cells on LB plates in the fridge for a couple of weeks at most-anerobic stabs last longer, a couple of months at least. For longer term storage you really need -80 or vapor phase liquid Nitrogen, or even lyophilization. Cells that are metabolicly active change over time - it's the nature of the beast! Tom
On Thursday, November 29, 2012 11:50:48 AM UTC-5, Cathal wrote: > 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 >
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