Technique / Molecular Biology / DNA analysis techniques / DNA southern blot


Sensitivity of DIG-Southern?



Sensitivity of DIG-Southern?

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 ... ...

Last update 08-Feb-2008, Rating of 1 votes.

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By Jen on 08-Feb-2008
I'm actually having a similar problem, but I'm looking for a single copy gene in a species of mosquito. I'm using the NEBlot Phototope Kit from NEB, and my positive control comes up right away, but getting anything from my samples is hit and miss. When I do get a signal, it is extremely faint. We are using 10ug of digested genomic DNA and up to a couple of ug of probe. Any ideas?
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