Dig-labelled DNA & chemiluminescent detection
verland at rockyj.rockefeller.edu
Fri Feb 28 19:59:41 EST 1992
ALSOBROOK at BIOMED.MED.YALE.EDU writes:
> it seems that some
>important details were left out of the article. For instance, the article
>claims "approximately 10 min" exposures on X-ray film for single copy
>genomic blots; the protocols say to start with a 60-min exposure, but
>since the light emission plateaus after 5-6 hours, at *that* point you
>can get a 10-15 min exposure.
I have spent a little time trying out chemiluminescent detection - I haven't
used Boehringer's kit, but I have used their substrate. In my experience,
the output does in fact increase over time - but that doesn't mean that you
can't go ahead and immediately put your filter on film. The limiting factor
was always the level of background from non-specific binding of my
detection reagents to the filter (but I was using biotin/streptavidin, not
digoxigenin). As a result, a 2 hour exposure was the longest I could use.
I would suggest that you go ahead and try some short exposures, beginning
as soon as your filter is ready - there's a good chance that you'll have
adequate signal, even though the output hasn't peaked yet. If that doesn't
work, you can just put the filter back on film, since the alkaline phosphatase
is still cranking out signal - and all you lose is a piece of film or two.
I found 32P to give me better results - but if they've licked the background
problem, chemiluminescence might do the job.
verland at rockvax.rockefeller.edu
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