Northern blot problems

Marek Treiman mat at biobase.dk
Tue Jun 10 04:34:39 EST 1997


Would greatly appreciate hints on the possible causes of our northern
blots problem:

We are trying to detect a medium-abundance message in RNA-extracted from
PC12 cells. We use
a biotinylated probe + ECL detection. We estimate having about 0.1
fmoles of mRNA of interest on the gel, and use about 1000 fmoles of the
probe.The probe and detection system work OK in tests on
southern blots. The probe is a double-stranded DNA. RNA (total 10
micrograms/lane) is run on formaldehyde gels and transferred to Hybond
N, with v.good efficiency. It is fixed by baking at 80 degrees C x 30
min.
 Hybridisation is at 42 deg.overnight in 50% formamide, SSCx5, SDS 1%.
We have no background on ECL-films, but 
no signal either!  WHY??
Tips greatly appreciated!
Best regards, Marek



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