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

The plates do fluoresce somewhat with blue or UV light, but it's a
blueish fluorescence. Bare plates have nothing like this vibrant orange
fluorescence.

Also, the fluorescence is limited to the colonies themselves, not the
area around them, suggesting it's not a kanamycin breakdown product.

I'm streaking out my old stocks of DH10B to see at what point could
contamination have occurred, and I'll streak on plain and Kan+ plates to
see the difference. So far, it does look like the stock I was using this
month has contamination built-in, possibly the older stocks are clear..
I'll have to incubate another few days and see.

On 30/11/12 05:33, Nathan McCorkle wrote:
> Does a concentrated solution of the kanamycn fluoresce? Maybe it
> degraded and only some bacteria have the right pump or something
>
> http://www.sciencedirect.com/science/article/pii/0378109790900028
>
> On Nov 30, 2012 12:24 AM, "Nathan McCorkle" <nmz787@gmail.com
> <mailto:nmz787@gmail.com>> wrote:
>
> Also it looks like you didn't select a colony for your exponential
> growth culture... maybe fridge mutants?
>
> On Nov 29, 2012 11:50 AM, "Cathal Garvey" <cathalgarvey@gmail.com
> <mailto:cathalgarvey@gmail.com>> 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
>
> --
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> twitter.com/onetruecathal <http://twitter.com/onetruecathal>
>
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