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

If it's straight contamination (which I'm taking as the most likely
answer), then it's not just the orange guys: I've also got "plain
looking" cells which look similarly like E.coli, and turn up on
negative and positive plates.

I'm currently running two plates from an old stock of DH10B: one
without selection, one with. If I get growth on the clear plate and
none on the Kan plate, I'll be happy, and will re-start the culture
from a colony.

You're probably right about the outgrowth: I knew some was necessary
after transformation with Kan and other bacteriocidal antibiotics, but
I was only giving them about 30 mins... and my incubator is only 30C.
Might want to leave them 1:30 hours next time: will be repeating
transformation next week hopefully.

Thanks for the advice guys!

On Fri 30 Nov 2012 15:11:08 GMT, Matt Lawes wrote:
> Maybe your DH10B stock isn't quite so.
> Perhaps a contaminant with low innate KanR is in the LB stock. I would
> make some plates with higher kan concentration .... 100, 150, ug/ml
> and see what kills the orange guys.
>
> I would also reisolate the e coli from your stock on plates without
> kan ..... looking for the not orange colonies - sounds like one more
> round beyond what you've done will give you a pure stock.
>
> Kanamycin also needs time ( an hour of outgrowth without Kan) for
> phenotypic expression after transformation of the plasmid. Didn't look
> at your protocol to see of you have that down.
>
> Best,
>
> >matt
>
> /Sent from my Verizon Wireless 4G LTE DROID/
>
>
> -----Original message-----
>
> *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>
> <http://www.indiebiotech.com>
> > twitter.com/onetruecathal <http://twitter.com/onetruecathal>
> >
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