Dig-labelled DNA & chemiluminescent detection

John Nash num208jn at MBDS.NRC.CA
Thu Feb 27 22:37:57 EST 1992


In article <01GH0W1OX5O0002PGW at BIOMED.MED.YALE.EDU>, ALSOBROOK at BIOMED.MED.YALE.EDU writes:
>Greetings, netters! How about some advice? If you have used (or tried to
>use) the Boehringer-Mannheim non-isotopic DNA detection methods, 
>specifically using the new Lumi-Phos chemiluminescent substrate, please
>drop a line to this newsgroup and share your experience with us! An
>article in the Jan '92 BioTechniques sung the praises of this method
>(of course, it was written by some B-M folks!); after purchasing a kit and
>reading the actual technical protocols included, 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. So yes, you can get short exposures, but
>first you have to wait!!  Any experience anyone else has had would be
>most informative!! Thanks for reading this.
>
>****************************************************************************
>* John Alsobrook                        |    Never ask a question whose    *
>* Child Study Center                    |    answer you are not prepared   *
>* Yale University School of Medicine    |             to hear.             *
>*--------------------------------------------------------------------------*
>*  expressed opinions are strictly my own, but you can have them for free! * 
>*                  bitnet: alsobrook at yalemed.bitnet                        *
>*               internet:alsobrook at biomed.med.yale.edu                     *
>*--------------------------------------------------------------------------*

I don't think you have to wait ;-)

What we do is... put a couple of ml of Lumpihos (or AMPPD) on a large
watch glass, and dip the colony/DNA side of the filter(s) in it until
the entire surface has been wet.  Then we drain the filter(s) on the
edge of the watch glass and seal them in Dazey heatseal bags.  We find
Ziplock bags inactivate the signal.  Then we incubate the bags in a 37
deg incubator for 15 - 30 min, and expose (Kodak X-Omat XAR5 film) 5 -
15 min, and develop.

I waited 2 hr the first few times, and got black filters, and was
ready to swear at BM!  I reduced the exposure time and it worked
better.

I generally do my hybridizations at 10 - 25 ng probe /ml hyb solution,
and if I have 50 or so filters, I do them in a Rubbermaid container,
with some Whatman paper on the top to stop the top few filters drying
out.  I've re-used the same probe 6 or 7 times over a six month
period.

Oh yes, we re-use the LumiPhos, too.  We put it in a scintillation
vial, store it at 4deg and re-use it until it runs out.

(We isn't the royal "we".. it means a couple of us around here do it a
similar way.  "I" means in my experience...)

cheers,  John.

Dr. John Nash,                       | 
Cell Systems Section,                | Email:
Institute for Biological Sciences,   | Preferred: John.Nash at NRC.CA
National Research Council of Canada, | or       : num208jn at mbds.nrc.ca
Ottawa, Ontario, Canada.             |
      ==>  Disclaimer:  All opinions are mine, not NRC's!  <==



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