hybridization of oligos to northern blots
BOUTILIERK at EM.AGR.CA
Wed Nov 22 13:49:46 EST 1995
I have hybridized oligo probes of 17 to 39 nt on both RNA gel
blots and dried agarose gels (for genomic DNA) with great success.
The probes were 5' end labeled with T4 polynucleotide kinase.
Unincorporated nt were removed using either NAC ion exchange
columns from BRL (for oligos shorter than 29 nt) or on sephadex
G-50 columns (for oligos 29 nt or longer). I prehybridize the
Northern blots in 5X SSPE, 5X Denhardt's, 0.5% SDS and 200 ug/ml
tRNA at 65 oC or Td-15oC (whatever is the lower temperature) for
at least 1 hour and then hybridize overnight (in the same
solution, at the same temperature) using 1, 000, 000 cpm probe/ml
hybridization solution. The final temperature and salt
concentration at which you wash your blots will depend on the
melting temp of your probe etc... I run a cold riboprobe on the
gel as a positive control.
I have also hybridized oligo probes directly to dried gels. In
this procedure the gels are denatured, neutralized and dried under
vacuum (no blotting). The oligo is hybridized directly to the
dried gel without any prehybridization. Long probes, as well as
non-radioactve detection methods can also be used. The advantage
of this method is (supposed to be) 5X more sensitive than when you
hybridize to transfers, although dried gels are not as durable as
nylon memebranes. This procedure works really well with genomic
DNA digests, but I didn't have much luck hybridizing oligos
directly to dried RNA gels (they come out black!). I run my RNA on
formaldehyde gels and maybe this has something to do with it i.e.
glyoxal or methyl mercury gels might give better results.
Look in Maniatis for general info on oligo hybridization and in
Gottlob-McHugh and Johnson (1991). Detection of a subfamily of
genes within the soybean nodulin-A-multigene family. Can. J. Bot.
69: 2663-2669 (and references therein) for specifics on probe
labelling, Northerns and dried-gel hybridization.
Contact me directly if you want more details, protocols or run
into any problems.
Plant Research Centre
Agriculture and Agri-Food Canada
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