Southern blot mystery

NJ njuni at poppy.ocn.ne.jp
Fri Jul 6 04:14:10 EST 2001


Probably, blotted genomic DNA worked as a blocking reagent so that the
blotted area had less background than the blank area (as you know, DNA is
used as a blocking reagent to reduce nonspecific absorption of a
hybridization probe into membrane). So my guess is that DNA was surely
transferred but the amount of target DNA, sensitivity, or S/N ratio were not
enough. If you add carrier DNA into hybridization buffer, which is not
included in the standard DIG hybridization buffer, such white bands might
become indistinguishable from blank area.

By the way, how much amount of genomic DNA did you blot? The biding capacity
of membrane to DNA/RNA would be no more than a few micro grams per slot. If
you overload, I imagine, DNA might pile up unstable on membrane, and happen
to peel off during the procedure, even after hybridized with a probe....
--
NJ

in article 20010705210907.6096.qmail at ww02.jatek.com, Joe Appelgryn at
appelgjj at sci.uovs.ac.za wrote on 7/6/01 6:09 AM:

> I've done a slot blot, using a MINIFOLD II system with different
> concentrations of genomic DNA and a purified fragment, the same as my probe,
> as a control.  The detection is by means of chemiluminescent methods, using
> DIG nonradioactive detection system from Roche Molecular Biochemicals (also
> Boerhinger-Mannheim).  The first time I did the slotblot you could see the
> bands perfectly.  When I repeated the blot with the same DNA, and other
> conditions, the result was unexpected.  The purified fragment, used as
> control, came out as a black band but the rest of the bands appear white on
> the x-ray film.  I was told that it is negative staining, but nobody could
> told me what the possible cause might be.  I also don't know if you could
> see it as a "positive" result, or was there simply no DNA transfered.
> I would appreciate it if somebody could help me.
> 
> 
> 
> Joe Appelgryn
> 
> Department of Botany and Genetics
> 
> University of the Free State
> 
> Bloemfontein
> 
> South Africa
> 
> 
> <http://www.biowww.net/forum/read.php?f=1&i=2827&t=2827>
> 




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