Help: GAPDH probing problem

Mikael Niku mniku at paju.oulu.fi
Thu Aug 21 05:02:04 EST 1997


I've been trying to do a control probing for a few northern
filters using a rat GAPDH DNA probe labelled with Pharmacia
nick system.
Every time the result is that large areas of the filter have
extremely high background, largely hiding the signal and
thus making use of the filters impossible. It's very difficult
if not impossible to wash the filters.
The background isn't even, covering only parts of the filter.

I've done several other hybridizations with the same
materials (except the DNA, of course) and equipment. Everything
has been fine, but now I can't make this one work.

For the GAPDH probing, I've used a standard prehybridization
solution in SSPE, containing 50 % formamide, and prehybridized
about 4 h before adding the probe, then hybridized overnight.
Everything has been in 42 C.

The GAPDH DNA was originally in a puc; now I tried cutting it
off. Still no success.

Any ideas welcome!

-- 
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+++ Mikael Niku   +++  E-mail: Mikael.Niku at oulu.fi +++++++++++++
+++++++++++++++++++++  http://www.student.oulu.fi/~mniku/ ++++++
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