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.
KG
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?
>
> --
> View this message in context:
>
http://www.nabble.com/Drosophila--In-situ-hybridization-problems-
> tp14648451p14648451.html
> Sent from the Bio.net - Dros mailing list archive at Nabble.com.
>
>
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