Help! Problem with Northern Blots!

Bob Feather bob129 at megsnet.com
Mon Apr 14 22:20:03 EST 1997


Joy Buckle <jbuckle at cs.mun.ca> wrote:

>
>I have been carrying out Northern blots for the last six months and the
>lab tech here has been doing them for 2 or 3 years.  Things up until now
>were fine but in the last month or so we have been producing blots that
>are literally half black (full of radioactivity) and half normal. For
>example, today I developed a membrane from a large gel.  The bottom lanes
>were perfect but the top half was simply black.  It was impossible to even
>determine where the lanes were. This is why it is so puzzling.  We are
>using nylon membranes and prehybridizing for 4 hours in Denhardt's, SDS,
>SSC and Salmon Sperm DNA.  This is being carried out in a hybridization
>oven @ 42 C in the bottles.
Based on your description, this sounds like:

a) Your film cassette is bad, or
b) The film you have been using was partially exposed to light,
c) Someone opened your cassette, or it was improperly closed.

To isolate the problem, first test the film. Take a piece of
undeveloped film and develop it to see if has been exposed to light.
Second, put fresh x-ray film in two different cartridges: one in the
cartridge that gave you a black top, another in a cartridge where this
problem has not occured. Of course, if this problem occured in film
from multiple cassettes, you can rule out cassette light leaks as a
factor.



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