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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