Spots on Northerns?

John R. Thompson jrt at home.com
Sat Mar 14 10:37:56 EST 1998


I've tested for nucleotide binding (control experiments are wonderful
things).  In 2X SSPE and higher, nucleotides don't bind to charged
nylon membrane (at least Nytran+ and GeneScreen brands).  In fact,
this makes a good quick method to test your incorporation as the
oligos do stick.  Consequently, I never purify my random primer
labeling although I do perform TLC to check the progress of the
reaction (PEI plates, working from memory, develop in 0.75M Ammonium
Acetate, oligos stay at origin,  Load the smallest sub microliter
amount you can pipet from a 50 uCi labeling and autorad for 10 min or
expose to a phosphorimager screen for as long as it takes to walk down
the hall to the reader).

Speculating, I suspect the dot-type noise  is due to small areas of
the membrane that did not wet properly.  Try carefully laying the
filter on the surface and watch the wetting proceed across uniformly
across the filter.  I find the membranes wet more easily and evenly in
formamide-containing buffers.

Regards,
John Thompson
Merck & Co.

bmorriso at red.seas.upenn.edu (Barclay Morrison III) wrote:

>What causes the small punctate dots on radioactive Northerns? Sometime I 
>get them and sometimes I don't. It does not appear to be a phenomenon 
>associated with background as I can get these dots on blots with low 
>background.
>
>I have heard that they might be caused by un-incorporated radionucleotide 
>in the probe. What are your thoughts?
>
>Thanks




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