Help spotty northern problem help

Ulrich Maier maieru at uni-muenster.de
Thu Aug 7 04:59:29 EST 1997


punk at leleand.stanford.edu wrote:

>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

I have seen the same on Southern and Nirthern blots. IMHO it is caused
by handling of the membran with powderd gloves or by high
concentrations of salts on the membran.

I guess you are blotting capillary with 20xSSC. Wash the membran after
transfer and crosslinking first 5min with 2xSSC, then with Aqua dest
before hybridization.

Hope this helps,

Uli Maier





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