from coating the membrane. Good blocking is necessary to
minimize background problems.
The NorthernMax and NorthernMax-Gly kits include
Hybridization Buffer, which can be used for both prehybridization and
hybridization. Although double-stranded DNA probes must be denatured prior
to use, RNA probes and single-stranded DNA probes can be diluted in a
small amount of ULTRAhyb and then added to the prehybridized blot. ULTRAhyb
can increase sensitivity up to 100-fold compared to other hybridization
solutions (Figure 2) by pushing hybridization to completion without increasing
background. As few as 10,000 molecules can be detected. Because ULTRAhyb
maximizes blot sensitivity, hybridization can be performed in just 2 hours
for many messages. The largest increase in sensitivity is seen using random-primed
DNA probes and ULTRAhyb. Under these conditions, signals are as intense
as those seen with RNA probes.
Figure 2. ULTRAhyb versus a standard hybridization
buffer using DNA and RNA probes. Identical
Northern blots of indicated amounts of mouse thymus total RNA were hybridized
overnight with radiolabeled StripAble DNA or RNA probe. The blots were
incubated with probe in either ULTRAhyb or standard hybridization buffer
as indicated. Standard low and high stringency SSC/SDS washes were used
to remove nonspecifically bound probe. Blots were exposed to film for
2.5 hours with a single intensifying screen.
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