Problem with inversed binding on a dot blot

Andreas Weber aweber at biolan.uni-koeln.de
Fri Aug 26 15:42:33 EST 1994


In article <dwhite-2608941328290001 at moe.fcs.uga.edu>, dwhite at hestia.fcs.uga.edu (doug white) says:
>
>   I recently ran across a strange problem on a dot blot I was running.
>The autoradiograph image was inverted.  The probe bound to everywhere on
>the membrane except the dots.  It's as if there where alot of non-specific
>binding with the membrane (Genescreen plus) and the dotted RNA prevented
>it. It was a commercial cDNA actin probe.  It was hybridized at 52 C in a
>phosphate buffer, containing 0.5M Na, 50 % formamide and 7 % SDS.  Does
>anyone have an idea what could have caused this? Thanks in advance.
>
>Doug White
>dwhite at fcs.uga.edu

Dear Doug,

this "negative" effect is often seen when you use to high a concentration 
of 32P-probe. For details see Anderson&Young, Quantitative filter hybridisation
in: nucleic acid hybridisation:a practical approach; irl press, Oxford, Washington DC

Hope this helps and good luck,

Andreas


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