Home / Molecular Biology / RNA transcriptional and post-transcriptional regulation / Northern blot hybridization
RNA Northern blot hybridization protocol
protocol
This is a standard northern blot hybridization protocol using radioactive labeled probe. The northern blot protocol includes details on total RNA sample preparation, agarose gel electrophoresis using MOPS running buffer, northern blotting membrane transfer pro ...
Weird Northern blot hybridization
troubleshooting
I was doing northern analysis with arabidopsis AOS gene. When I used the
5' fragment of the gene as a probe (from the EST), it worked
well. However, if I used the 3' fragment as a probe (from PCR), it always
hybridized to all the rRNA (including 26S, ...
Formaldehyde pH for Northern blot
troubleshooting
We've been having trouble with our RNA degrading during Northern's.
We've discovered the culprit is the formaldehyde and particularly the
denaturing step (we denatured with formaldehyde in the sample). The pH
of our formaldehyde was (4.4) which we had ...
(Curr Protoc Mol Biol. 2004 Sep;Chapter 4:Unit 4.9.)
Brown T, Mackey K, Du T.
University of Manchester Institute of Science and Technology, Manchester, United Kingdom.
Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern blotting differs from Southern blotting largely in the initial gel fractionation step. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The Basic Protocol describes blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. Alternate Protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a Support Protocol.
Last update: 
07-Feb-2012 02:38 am
