Re: [DIYbio] Re: Kanamycin Stability, & Mysterious Orange Colonies

Sorry, the concentration of the stock was mg/ml, the final concentration
was ug/ml.

On 29/11/12 22:46, Xabier Vázquez Campos wrote:
> What is the final Kan concentration? Because 50 ug/mL for a stock is
> quite low.
>
> El viernes, 30 de noviembre de 2012 03:50:48 UTC+11, Cathal escribió:
>
> 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
> <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 <http://www.indiebiotech.com>
> twitter.com/onetruecathal <http://twitter.com/onetruecathal>
>
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