Help! No signal in southern blot - troubleshooting (Aug/05/2008 )

Hello everybody!

I need some help with my southern blot.
I've already tested my putative transgenic plants via pcr. Afterwards I wanted to make a southern blot in order to see the copy numbers. But somehow this isn't working. I tried it several time already. The problem is that I get no signals from my plants. I included a positiv control (the plasmid which is carrying the desired gene) and a dig-labeled marker. I get signal from both of it but no signals frommy plants. So I quess that the transfer and the probe should be allright. But still there is NO signal. Do you have any suggestions why there ist no signal? In the following I will give you the protocol that I used. Maybe this helps:

Isolation of the genomic DNA with CTAB
Digestion of genomic DNA and the control plasmid either with EcoRI or XbaI in 300 µl
Precipitation of the digested DNA with Ethanol
Agarose Gelelectrophoresis on an 0,8% (w/v) agarose gel, 100 V, 2.5 h without EtBr
EtBr Staining of the gel
Denaturation of the DNA 2 times with 0.5 M NaOH for 15 min
Depurination 2 times with 0.2 M HCL for 15 min
Neutralization with neutralization buffer (0.5 M Tris-HCl, pH=7,5 und 1,5 M NaCl) 3 times for 10 min
Transfer of DNA via capillary transfer method over night, 10x SSC as denaturation solution
UV crosslinking for 15 min on a UV table
Prehybridization: 30 ml DIG Easy Hyb for 2 h at 42°C
Hydridization: 30 ml DIG Easy Hyb plus 3 µl probe over night at 42°C
Washing: 2 times 2x SSC+0.1% SDS for 5 min and 2 times 0.5xSSC+0,1% SSC at 65°C for 15 min
Detection:
1h in 1x blocking reagent
30 min in antibody solutuion (anti-DIG-AP 1:10000 in 1x blocking reagent)
3 times 1 x Maleic buffer for 10 min
2 min in detection buffer (0.1 M DEA)
CDP-Star 1:100 in detection buffer, incubation on Membran for 5 min
Exposure on film for 1 h, 2 h or over night

I am very desperate. So I would be glad if someone helps me!

Thank you!

lieschen

It looks like the procedure is fine!

As you are getting no signal in the plant samples, perhaps there is only one copy, and you need to expose the films for longer. I suggest titrating the plant DNA out as well, start off with what you are using at the moment and work up in amount added to see if you can get some signal. You may need several ug for this. Remember, the signal strength is proportional to the percent the target makes up of the DNA, with a plasmid the signal may be as much as half of the total DNA, but in real life clones the target will to be much, much less.

Hello bob1!

Thanks for the reply. I already exposed the film for 12 hours and more. And there was no change. I also tried to increase the amount of DNA. The digestion was carried out in 300 µl. Afterward I precipitated the DNA. I already loaded several µg of DNA on the gele. But there is still no signal. The next thing that I will try is to detect a standard gene like actin. I hope this will give me more information about the problem. Anyway, thanks for your suggestions!

lieschen