[Drosophila] Drosophila- In situ hybridization problems
(by yehuditmi from gmail.com)
Sun Jan 6 08:27:00 EST 2008
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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