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

From: Cathal Garvey <cathalgarvey@gmail.com>
To: "diybio@googlegroups.com" <diybio@googlegroups.com>
Cc: "Xabier Vázquez Campos" <xvazquezc@gmail.com>, "methods@net.bio.net" <methods@net.bio.net>, "methods@magpie.bio.indiana.edu" <methods@magpie.bio.indiana.edu>
Sent: Fri, Nov 30, 2012 10:01:44 EST
Subject: 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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