Help spotty northern problem help

harald at mol.univie.ac.at harald at mol.univie.ac.at
Thu Aug 7 11:41:35 EST 1997


In article <33E76F50.3D1E at leleand.stanford.edu>, punk at leleand.stanford.edu 
says...
>
>Hi, I am doing a lot of northerns on cellular RNA and having a problem
>with spots on my autorads. These spots are small and round. The
>hybridization signal is good, no crosshyb to rRNA and the general
>background is low except for the spots. I am using  zeta-probe membrane
>from biorad and have changed lots which didn't help
>
>- I am using the .5M NaPO4  7%SDS  hyb buffer. It used to work well no
>spots and this is what is recommended for the zetaprobe.  I have switch
>the solutions and filtered them but still the spots are present.
>
>- I am using random primed probes and a hybridization oven. The probe is
>cleaned up using qiagen spin column 
>
>If any body has any suggestions on what might be causing these spots
>please post to this group I check it regulary Thanks alot gregg


Hi!


I had a similar problem when doing Northern blots. However, I was told by 
a specialist that this is caused due to partial drying of the blot: the 
hot probe apparently gets linked to the membrane and cannot be stripped 
later. Therefore, my suggestion is to keep your blots nice and humid (some 
prefer acually wet) in order to avoid the spots. Please mail me if I am 
right or not!


good luck, Harald




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