Digoxygenin End labelling

Fri May 12 09:07:47 EST 1995

In article <A.C.Hilton-1205951244440001 at bcs88.bham.ac.uk> A.C.Hilton at bham.ac.uk (Wiggy) writes:
>Subject: Digoxygenin End labelling

>I am considering end-labelling a pBR322 hinf1 digest with DIG dUTP so I
>can get a marker for Southern blotting that I am doing.  I am concerned
>that the single (maximum 2) incorporated DIG-dUTP will be insufficient to
>give a detectable flourescent signal. Does anyone have any experience of
>end labelling with such non-isotopic methods at these low levels?
>I am not opposed to labelling with 35S or 32P but the development times on
>film are vastly different,  only 30 minutes with the DIG. 
>Are there any commercially available DIG markers?
>I already have a number of DIG labelled primers so a DIG marker would be 
great.>Thnks for your time,

It may strongly depend on whether you are Southern blotting prokaryotic vs. 
eukaryotic genomic DNA.  We've used biotinylated oligo probes for bacterial 
Southerns (single biotin label) of single copy genes and have had no problems 
(actually, beautiful blots).  I guess with eukaryotic Southerns, you would 
have to deal with up to 1,000-fold more extraneous DNA because of the larger 
genome size and therefore probe sensitivity becomes more of an issue.

-Regards, Peter
*  Peter M. Muriana, Ph.D.             317-494-8284   TEL            *
*  Dept. of Food Science               317-494-7953   FAX            *
*  Purdue University                   murianap at foodsci.purdue.edu   *
*  Smith Hall                                                        *
*  W. Lafayette, IN  47907-1160                                      *

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