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.
                            Be lucky,
                                        Marat Gorivodsky
                                        arkadyt at post.tau.ac.il



More information about the Methods mailing list