Microwaving for mRNA hybridisation
Bryan L. Ford
fordb at bcc.orst.edu
Fri Jul 10 11:00:19 EST 1998
bill.kalionis at flinders.edu.au wrote:
> We have been microwaving tissue culture cells (JEG-3 and JAR) in
> order to carry mRNA in situ hybridisation. we carry out the
> recommended 15-20 min of microwaving. When we hybridize Digoxigenin
> labeled oligo probes there is a very strong signal but we get this
> regardless of which oligo probe we use i.e. sense, antisense,
> completely unrelated. This only happens when we add probes, if we dont
> add probes there is no signal hence the problem doesnt seem to be
> coming from the secondary reagents.
Microwave heating is quite subject to variability depending on the
geometry and size of the surface/volume to be heated. Additionally,
different microwave ovens have quite diverse distributions of energy.
Check the actual temperature of the solution after microwaving using a
digital thermometer with a low mass probe, then make appropriate
adjustments in your protocol. For highest reliability you might consider
switching to some other means of heating, such as conventional heat
blocks, baths or incubators.
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