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GUS histochemistry assay


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#1 Karmaiko_UOIT

Karmaiko_UOIT

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Posted 25 June 2010 - 06:49 AM

Hi all, as this is my first post I thought I'd give an introduction and then describe what I'm doing/the problem!
I'm an undergrad at the University of Ontario Institute of Technology located at 2000 Simcoe St. North in Oshawa Ontario. I'm researching Arabidopsis thaliana.

The protocol I'm running is using two reporter genes, cyc::GUS and DR5::GUS (DR5 is an auxin inducible gene), and after growing the seeds on MS media for 6 days I am trying to obtain some staining results (as you've probably guessed from the title).
I've prepared pH 7 Gomori buffer, X-gluc substrate solution: ~1mg of X-gluc dissolved using 99.9% Methanol (not N,N-dimethylfermamide), 1mL 2x Gomori buffer, 20 uL Potassium Ferricyanide (III) and Hexacyanoferrate (II) at 0.1M, and 10 uL 10% w/v Triton X-100 all in 0.85mL dH2O
The seedlings were washed in the X-gluc substrate and left to incubate for 18 hours but there's no staining/colour change occuring! The protocol I'm using (obtained here http://microscopy.ta..._in_plants.pdf) has some slight modifications - no vacuum infiltration or fixative were used. The seedlings were incubated in darkness at room temperature and it was expected that they would produce a dark blue colour.

So my questions is of course what's the mistake I'm making to not see colour change??

thanks
Karm

#2 Karmaiko_UOIT

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Posted 25 June 2010 - 07:30 AM

Nevermind! I've diagnosed the problem and come up with a solution, the incubation worked but not in enough plants, I'm rerunning the procedure under more stringent conditions!




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