Nonradioactive Northern Blots

Martin Offterdinger a8803349 at unet.univie.ac.at
Thu Jul 10 03:11:35 EST 1997


On 1 Jul 1997 10:00:38 -0700, cxi3 at PSU.EDU (Chris Ingle) wrote:

>Hi All!
>
>Has anyone out there had any success with N. blots and
>nonradioactively-labeled probes?  I have been trying to probe my blot
>with a DIG-labeled probe, and visualize using chemiluminescence, with no
>success.  My probe is efficiently labeled, but after I hybridize (using
>Clontech's ExpressHyb), wash, and expose, nothing.  Well, except a
>little bit of a schmear on the blot, but I know my probe is highly
>specific, so I think that's just background.  Our lab is currently
>waiting to be cleared for radioisotopes, so until then I have to use
>other means.  Any ideas/advice? Please e-mail me directly  rather than
>posting to the newsgroup, if possible.
>
>Thanks,
>Chris Ingle
>Penn State University
>Dept. of Veterinary Science
>115 Henning Bldg
>University Park, PA  16802
>(814) 863-8527
>cxi3 at psu.edu
>
Dear Chris!
We have used the DIG system with varying success: The best way seemed
to be using DIG labelled riboprobes at a hybridisation temperature of
65° centigrade. We had no success using cDNA and even with riboprobes
the system does not seem to be very sensitive.
I hope that this doesnt sound too discouraging !
martin



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