need HELP with non-radioactive Northern hybs

John Nash nash at nrcbsa.bio.nrc.ca
Fri Mar 11 21:17:42 EST 1994


In article <aquilla.1113810678B at sadye.emba.uvm.edu>,
Tracy Aquilla <aquilla at salus.med.uvm.edu> wrote:
>Greetings gene jockeys,
>  I am interested in using non-radioactive labeling for quantitative
>Northern blot analysis, and I need help deciding which system to use. Any
>comments from experienced users of such products would be sincerely
>appreciated. I need high sensitivity for this assay, and I prefer to use
>oligonucleotide (ssDNA) probes.
>  I think the LightSmith (Promega) and Aces (BRL) type of approach seems to
>be the best idea I have seen so far, but I am reluctant to put an
>AP-conjugated oligonucleotide through the rigors of a stringent
>hybridization protocol, for fear that it will inactivate the enzyme. Also,
>what about HPR-conjugated oligos? Has anyone used these or similar products
>with success?

How about labelling your oligo with digoxigenin (with DIG-11-dUTP or
DIG-11-ddUTP from Boehringer Mannheim), do the hybridization, then use
an AP-conjugated-anti-DIG to detect the DIG-oligo, using a
light-emitting AP substrate (Lumiphos or AMPPD).  I do this all the
time with "semi-quantitative" DNA dot-blots, and a colleague does this
with colony lifts.

[Q: "semi-quantitative" What does he mean??? A: Eyeballing the results
on the autorad because I haven't got around to using the luminometer
yet.]

-- 
John Nash                           (nash at nrcbsa.bio.nrc.ca)
Institute for Biological Sciences,  National Research Council of Canada,
                 Yet another Aussie-in-exile ;-)
      *** Disclaimer:  All opinions are mine, not NRC's! ***



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