Help! Problem with Northern Blots!

Joy Buckle jbuckle at cs.mun.ca
Wed Apr 9 12:42:31 EST 1997


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.

Only one membrane is contained in each bottle. We then hybridize overnight
with the DS probe @ 42 C again.  The one thing I did notice this time was
that when I rolled the membrane and the nylon mesh into a tight spiral to
place it into the bottle, the part of the membrane that turned out to be
black was the part that was directly exposed to the solutions (i.e. facing
the interior of the bottle).  

This was working fine before using all the same recipes for the solutions
used.  Anyway have any ideas what could be wrong?  We've asked evryone
here in the dept but no one has seen or heard anything like this before.
We even contacted Mandel, the producers of NYTRAN- the nylom membrane that
we are using)  but to no avail.


Any suggestions?   Please email jbuckle at ganymede.cs.mun.ca

Thanking you in advance,
Joy




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