[Drosophila] Drosophila- In situ hybridization problems
(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, weve had serious problems with high background, and some
> problems with weak signal. After examing all components, we see that
> most significant factor is the enzyme used for PCR! Most suprisingly,
> Polymerase from Roche gives us background problems that are often the
> and better results (but not consistently good) with enzymes like
> polymerase from NutriCyte (Buffalo, NY) and others. This is all in a
> in which were: using Roche anti-DIG; pre-absorbing the Antibody with
> embryos; and blocking by methods that previously worked well. The
> problems are not sequence specific, and arise with all probes.
> Does anyone have suggestions about this problem? Or any general
> for improved ways of reducing background?
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