RNA blot question
Julie Roberts
jroberts at rascal.med.harvard.edu
Fri Jul 26 15:10:26 EST 1996
We are learning the processes involved with total RNA extraction from
mice and making northern blots with this RNA. The RNA looks to be high
quality on formaldehyde gels and seems to be transferring well. We bake
the blots under vacuum at 80 degrees for an hour. When we probed the
first blot with a generic probe it seemed to work beautifully. We used
hybridization temperatures around 68 degrees and later read that this
may degrade the RNA and hybridization at 42 in the presence of formamide
was kinder. We then attempted to use more specific mouse probes
constructed in our lab using PCR and then began the trouble.
One blot in particular looks as though it has a reverse hybridization
pattern in which the probe has hybridized to everything but the RNA
bands. Has anyone seen anything like this before? Can anyone explain
this hybridization pattern?
Thanks,
Julie Roberts
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