Nonradioactive Northern Blots
Dr. A. Torchinsky
arkadyt at plato.tau.ac.il
Fri Aug 1 09:14:48 EST 1997
Eric Lader wrote:
> On Thu, 10 Jul 1997 08:11:35 GMT, a8803349 at unet.univie.ac.at (Martin
> Offterdinger) wrote:
> >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.
> We've had several people of late calling tech service with this
> problem. We routinely use biotin labeled probes, but I inderstand why
> you want to use dig (using it for in situs, right?)
> Try the CDP star chem substrate from Tropix.
Dig-labeled RNA probes are working perfect in my hands for RNase
protection assay. I have used subsequent conditions : 45 C for ON
hybridization, 1 hours digest at RT with 16U of RNase ONE (Promega) and
CSPD substrate from Tropix.
arkadyt at post.tau.ac.il
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