Sensitivity of DIG-Southern?

[Dr. Klaus Eimert]

Dear netters,

I was trying to set up a non-radioactive Southern to confirm transgenic
plants. 
I used a DIG-labelled NPTII probe (PCR DIG probe synthesis kit from
Boehringer Mannheim; 2 µl probe per ml hybridization buffer) and 10 µg
of digested genomic arabidopsis DNA. 
Labelling, transfer and hybridization (O/N) went well (positive control
with plasmid). I didn't detect any signal for my positive plant control
(from the ABRC stock center), though - even after 4 hours color
development.
My questions: Did anybody use DIG-labelled probes to detect single copy
genes with color (AP and NBT/BCIP) in plants - what is your experience?
Does longer hybridization and/or color development improve sensitivity?
Should I use more DNA? Are there any tricks or hints?
Any suggestion is appreciated!
Thanx in advance!  

Regards,

Klaus

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Dr. Klaus Eimert
Dept. of Botany
State Research Institute Geisenheim	Phone:    + 49 6722 502 469
Von-Lade-Str. 3				Fax:      + 49 6722 502 460
D-65366 Geisenheim			E-Mail:   eimert at web.de
Germany					URL: www.mnd.fh-wiesbaden.de/fag/bio/bo/ebostart.html
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