non-radioactive DNA detection

Ed Rybicki ED at micro.uct.ac.za
Wed Dec 9 03:55:11 EST 1992


From:         "Ed Rybicki"  <ED at micro.uct.ac.za>
Date:         9 Dec 92 10:59:47 SAST-2
Subject:      Re: Non-radioactive detection systems
Reply-to:     ed at micro.uct.ac.za
Priority:     normal
X-mailer:     Pegasus Mail v2.3 (R4).

Dear David:

We switched to the Boehringer DIG kit some years ago, as I had a lab full
of people who really didn't want to work with radioactivity (young, of
female persuasion, child-bearing age, etc).  We have had nothing but good
experiences with it.  The kit seems expensive at first sight, but our cost-
and-buy lady worked it out as being cheaper than using (say) the
equivalent 32P kit, because (main reasons):

1) The probes, once labelled, last for EVER;

2) No radiaactives disposal or exposure problems.

Additionally, you can label up PCR products to high activity simply by
adding DIG-11-dUTP-containing nucleotide mix either from the kit, or from
a mix you make up yourself (from Promega nucs. and B-M DIG-dUTP) (for Jim
Graham, who doesn't like kits 8-)  ), and PCRing away normally.  Try that
with radioactivity - then explain to everyone who uses the machine after
you that no, it isn't hot...!

The detection kits (or make 'em up yourself, Jim) use either NBT-BCIP (or
other) insoluble dye product, OR use the proprietary (or make it up
yourself, Jim!!) TROPIX or Boehringer AMPPD chemiluminescence kit (see also
recent post on DIG-Northerns), which work VERY well, and give detection
close to the 32P limit and much quicker, in my experience (hours instead
of days).  You can also clean off probes, re-probe, re-expose, etc.

And no, I don't work for Boehringer, I just shamelessly solicit free
samples from them....

  ____________________________________________________________________
 | Ed Rybicki, PhD             |     "Emancipate yourselves from      |
 | (ed at micro.uct.ac.za)        |            Mental Slavery            |
 | Dept Microbiology           |          None but ourselves          |
 | University of Cape Town     |         Can free our minds...."      |
 | Private Bag, Rondebosch     |                                      |
 | 7700, South Africa          |             - Bob Marley             |
 | fax: 27-21-650 4023         |          (Redemption Songs)          |
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  ____________________________________________________________________
 | Ed Rybicki, PhD             |     "Emancipate yourselves from      |
 | (ed at micro.uct.ac.za)        |            Mental Slavery            |
 | Dept Microbiology           |          None but ourselves          |
 | University of Cape Town     |         Can free our minds...."      |
 | Private Bag, Rondebosch     |                                      |
 | 7700, South Africa          |             - Bob Marley             |
 | fax: 27-21-650 4023         |          (Redemption Songs)          |
  --------------------------------------------------------------------






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