Dig-labelled DNA & chemiluminescent detection

Cecil Chen LabMed cecil at hiv.med.umn.edu
Fri Feb 28 18:23:37 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.             *
>*--------------------------------------------------------------------------*

i have used dig-labeled DNA and oligos on dot and southern blots containing
viral DNA fragments (but not genomic blots for single copy genes) followed
by lumiphos treatment. yes, the blots turned black after ~15' exposure.
my problem has been high background though, and even using those conditions
described in the biotechniques article (in january 1992 issue), the
background can still be a problem sometimes. and i have to use 3-4 sheets of
x-ray films sometimes to figure out the proper exposure time.  
my advice is to use lumiphos in situations where sensitivity is
a must (e.g. genomic blots for single copy genes, or dig-labeled oligo  
probes).  in most situations, i think the BCIP/XP system is adequate.

cecil chen
cecil at microbe.med.umn.edu



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