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Help! Campylobacter Southern Blotting problems - can't get any signal from blot, should i try a different kit? (Apr/16/2008 )

Hi everyone,

Thanks for reading this, I hope someone has some answers for me because I've been repeating this blot for months now!
I'm trying to blot Campylobacter jejuni genomic DNA for a specific gene and even my positive control isn't lighting up.
I digest ~5ug of DNA for 4 hours with Hind III then run slowly on a 1% gel, digest looks nice and healthy.
I then transfer to a nitrocellulose membrane using a vacuum set up and the following washes:

  1. 10 minutes with depurination solution
  2. 10 minutes with denaturing solution
  3. 10 minutes with neutralizing solution
  4. then finally 1hr with 20XSSC
I use a cross-linker to bind DNA to membrane.
Probe is made using a 650bp cleaned up PCR product using Amersham's Gene Images AlkPhos Direct Labelling and Detection system.
Overnight hybridization is at 55 degrees C.
On day 2 I wash with primary and secondary wash buffers before developing blot with photographic film (manually with trays of developer and fixer).
I know all my kit is working OK because I did a Salmonella plasmid slot blot and that lit up brilliantly.

I've noticed a lot of people on here use DIG, is that better than Amersham's or cheaper/more easily available?

Any advice welcome as I'm running out of ideas!
Thanks again,

Lea. wacko.gif

-lea peake-

Dig is more selective (there are other things that can exhibit Alkaline phosphatase activity). This is likely not your problem.
I'd recommend approaching this from the end. First, take your probe and spot serial dilutions of your probe onto nitrocellulose. Determine whether you can detect the probe with your detection system, and at what dilution. Since you have a working Salmonella probe, do this with that probe as well, and compare their sensitivities.

Do you have a plasmid or other DNA sequence containing what you hope to visualize? If not, genomic DNA might do. If so, spot serial dilutions of the DNA onto nitrocellulose, crosslink, and try hybridizing your probe against the dilutions. Again, since you have a working example, do a parallel experiment with your working example. Determine whether you can see the DNA you are analyzing with your probe, and at what sensitivity.

Only when all of this is working do you bother to do a gel and blot. I'd recommend that you continue to spot serial dilutions (of both probe and target DNA) onto your blot as a control for hybridization and probe activity.