Help: low incorporation of P32 or DIG in probe.

[Marc Champagne]

I'd like some input about this problem:

I'm working on a gene which spans at least 16 kb, which has been subcloned into
3 fragments of 3, 6 and 7 kb.  When i make probes for southerns/northerns,I get
good incorporation when i use the 3 and 7 kb pieces (or parts of these), but i
can't get good incorporation in the 6 kb part, even if i use various
subfragments, or pcr products.  I've used P32 (following the Prime-It 
kit protocol, or the Maniatis Klenow protocol, both with random hexamers) or
with the B-M DIG kit. Controls work fine.

Any idea on how to troubleshoot?
Any other approaches i should try?  How good are southerns/northern using 
end-labelled primers?

Thanks,
m.champagne at cellbio.duke.edu

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