NON_RADIOACTIVE NORTHERNS- personal summary

Euan R. Taylor etaylor at ccu.umanitoba.ca
Wed Dec 16 16:37:22 EST 1992


I just conducted a short informal survey of DNA DIG users- based on this
80% of users think DIG-DNA probes are utterly unreliable.
In our experience there is no difference in reliability between a DNA
target and an RNA target. The bulk of the replies I have received indicate
that DIG-RNA PROBES are much more reliable.
Whilst radiation is expensive and unpleasant we have returned to it rather
than a system which has failed to provide us with any consistent results.
I  would note that we have no problems doing the same exact experiments
with 32p. Furthermore the consistency of signal strengths with DIG is
"pathetic" for instance - if we do a series of DNA dilutions and probe
with DIG-DNA there is no consistent gradient of signal intensities
ie. signal strength is not proportional to target concentration. When the
exact same dilutions are applied to a blot and hybridised to 32p we get a
perfect intensity series (same DNA, same manifold, same membrane, same
tubes, same blot, done in duplicate.)
Now I am not saying that this system "never works" clearly some people
like it, however my impression is that it can be very unreliable, and in our
case has involved a considerable expenditure of time and effort in a
futile attempt to get even the most basic and simple controls to work
reliably. 
Our problems are not related to the blotting side of the procedure because
we can re-probe unsuccessful DIG blots using a 32p probe (made from the
same DNA sample)  and get perfect, reproducible, interpretable results,
with no problem.
I just think that anyone who is thinking of moving over to a
non-radioactive system would be well advised to insist on some samples to
try before they start laying out batches of $500 and days and days of
time, on using the system.

Euan Taylor
etaylor at ccu.umanitoba.ca

The above opinions are my own and are nothing to do with, and do not
necessarily represent those of my institution



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