I have been trying Southern with DNA probes (DIG labeled), but my blots are unsuccessful. Is it worth trying with riboprobes, dig labeled again? Can someone provide a protocol which one has applied with success?
Probably not since your targets are limited so if you try more probes would not help much. In addition, the riboprobes are less stable and cost more, and you may increase the background instead. The best option is try to use the TSA technique to amplify your target to 100-1000x. Still use your DIG-probe then amplify by applying appropriate TSA reagent against the DIG. Those kids are commercial available and you can even get the trial size for 25 reaction to save some money while testing. The successful rate is instant if you get the all procedure right. Good luck! WUX:)
Tyramide Signal Amplification (TSA) Systems from PerkinElmer -
I had good results using DNA probes for southern blots (on Arabidopsis DNA). Do you check the labeling efficiency of your probes using the test strips? In case you have problems this is recommended. Not only can you check the quality of your probes, but also the solutions you use for the detection.
I agree with Eric. The key for successful Southerb blotting is how good your probe labeling is and how much DNA you have on the membrane. It may be helpful to purify the labeled probe (and it should be routinely done) to get rid of the free/unlabeled probe before usage. if possible, always use a positive control to make sure the probes is ok.