Help - Spotted Blots

[Robert Cobuzzi]

We are having an intermittent problem (thus the difficulty in determining the
cause) with our hybridized blots:  The signal(s) on the blots are spotted;
it is possible to see the pattern whether it be a simple band or a complex
2-D replication gel, but the pattern itself is spotted as though not all of the
DNA on the blot were being hybridized.  Put another way, it looks as though 
the blots are an abstraction (like a Seurat pointelism-type painting) of what
a normal blot should look like.

The experimental conditions are as follows:

Agarose gels are Southern blotted to Hybond-N nylon (the unchrarged material),
that has been pre-wetted, by downward capillary transfer with 10x SSC.  After
2 h transfer, the blots are air dried and cross-linked using a Stratagene
UV crosslinker at the default setting (approx. 0.12 joules).  Blots are
prehybed at 65 C in a Hybaid oven with a prehyb containing SSC, Denhardt's
reagent, SDS, and sodium pyrophosphate for 1-2 h.  Random primed probe is 
prepared using Ambion's DECAprime II kit (sa > 10e9), and hybed to blot for
16-18 h at 65 C.  Hybed blots are washed 3 x 2 min. by hand agitation of 
Hybaid bottles, then 3 x 20 min at 65 C in the Hybaid oven with a wash buffer
that is 0.2% SDS, 0.1% sodium pyrophosphate and 0.5x SSC.

Blots are exposed either to Molecular Dynamics phosphor screen or XAR film.
Either way the blots are exposed we still see spots on our blots intermittently
(ie, if the expt. is spotty then it doesn't matter if we expose to film or
phosphor).

Anyhow, if anyone has any ideas about cause or solutions, please email me:
cobuzzi at ubvms.cc.buffalo.edu or cobuzzi at sc3101.med.buffalo.edu.

Thanks,
Bob Cobuzzi