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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