[Drosophila] Drosophila- In situ hybridization problems

Kathleen Gajewski via dros%40net.bio.net (by sa08383 from wotan.mdacc.tmc.edu)
Tue Jan 8 17:38:00 EST 2008

A number of years back I had problems with staining that were caused by  
using Roche anti-DIG Ab that was purchased separately (as opposed to  
coming as part of a transcription kit).  I don't know it that's still a  
problem, but it may be worth looking into.


On Jan 6, 2008, at 7:27 AM, yehudit wrote:

> Our lab has been doing whole mount in-situ hybridizations on Drosophila
> embryos using random-primed or PCR-produced DNA DIG probes for years.  
> In the
> last year, we’ve had serious problems with high background, and some
> problems with weak signal. After examing all components, we see that  
> the
> most significant factor is the enzyme used for PCR! Most suprisingly,  
> using
> Polymerase from Roche gives us background problems that are often the  
> worst,
> and better results (but not consistently good) with enzymes like  
> Super-Nova
> polymerase from NutriCyte (Buffalo, NY) and others. This is all in a  
> system
> in which we’re: using Roche anti-DIG; pre-absorbing the Antibody with
> embryos; and blocking by methods that previously worked well. The  
> background
> problems are not sequence specific, and arise with all probes.
> Does anyone have suggestions about this problem? Or any general  
> suggestions
> for improved ways of reducing background?
> --  
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> tp14648451p14648451.html
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