ECL for colony hybridization

Pete Muriana MURIANAP at FOODSCI.PURDUE.EDU
Sat Oct 22 09:25:53 EST 1994


In article <Pine.SUN.3.90.941021142929.9254A-100000 at chip.ucdavis.edu> Rinkei Ko <ez034381 at chip.ucdavis.edu> writes:

>I routinly use ECL non-radioactive DNA labeling system (Amersham) for 
>Southern. Recently, I used same system for colony hybridization in order to 
>clone a gene of interest. Instead of getting specific signals for the colony 
>which has right plasmid, I got signals on all the colonies (False-positive).
>I wonder if this system is not suitable for colony hybridization because of
>non-specific protein-protein interaction, or washing condition was not 
>stringent enough to avoid non-specific bindings.
>Rinkei Ko: rko at chip.ucdavis.edu

Rinkei,
We use Tropix's Southern Light Chemiluminescent Detection Kit and also 
observed what you've described above.  Our kit is based on a 
biotin:avidin-alkaline phosphatase recognition and a recent add by Schliecher 
& Schuell which uses a similar system indicated that background can be reduced 
in colony lifts by incubating the filters with a proteinase-K solution, then 
wiping, yes wiping !, them with tissue/SDS solution.

We haven't tried this yet to see how effective it is, but if anyone else gets 
around problems with DNA probing of colony lifts with biotin-labelled probes, 
PLEASE POST!
-Regards, Peter
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*  Peter M. Muriana, Ph.D.             317-494-8284   TEL            *
*  Dept. of Food Science               317-494-7953   FAX            *
*  Purdue University                   murianap at foodsci.purdue.edu   *
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