colony screening with Amersham ECL (RPN 3000 and 3004)

afc at gnv.ifas.ufl.edu afc at gnv.ifas.ufl.edu
Thu Dec 17 09:12:57 EST 1992


In article <1992Dec16.151837 at genethon.fr>, bouchier at genethon.fr (Christiane Bouchier) writes:
> 
> A week ago I asked whether anyone had ever carried out tests for screening
> colonies  with Amersham ECL direct system. So far I received no suggestions
> that could help me overcome the problem we have of detecting peroxydase 
> specific hybridization because of a high background.
> Once again thanks for your help !

This may not have anything to do with your problem, but since no one else
has responded:

We tried using various nonradioactive detection kits with probes hybridized
to squashed mosquitoes.  We got very high backgrounds with every one that
we tried.  We then tested just the reaction reagents with the squashes (no
probe).  Again, we got very high background.  Obvious conclusion: insects
have peroxidases and alkaline phosphatases that survive NaOH, baking, and
hybridization.  Maybe you have the same problem with bacteria, since colony
lifts are full of protein.

We were able to eliminate this background by treatment with protease.  We
used a laundry stain remover which is about 500000 times cheaper than 
proteinase K (your prices may vary).  Details are upcoming in a note by
Johnson et al. in BioTechniques in March.

Andrew Cockburn
USDA

> Christiane Bouchier
> Genethon
> 1, rue de l'Internationale
> 91 000 EVRY
> FRANCE
> Tel : 19.33.1.69.47.29.66
> email : bouchier at genethon.fr



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