I am trying to confirm the presence of a putative 8.5 kb insertion in a Drosophila mutant, by PCR and/or Southern Blotting. I use almost 10 ug DNA for each lane, and specific probes(812 bp) which are DIG-labelled. I have tried countless number of times for a long time now, but my blots dont work. The blots are either clean or totally dark.
A number of things I can suggest:
1. Increase your DNA in the lane. While 10 ug may seem quite large, 15 or 20 ug might be necessary. Make sure to run the gel slowly for these higher concentrations.
2. Check your probe concentration. There can be a fine line between a blot with no signal and a blot that is completely exposed. Try increasing or decreasing probe concentration in response to how the blots look; if they are too dark, reduce the probe concentration, and if they are showing nothing, increase the probe concentration.
Additionally, make sure you are exposing your blots for the right amount of time; it can sometimes take overnight exposures to get a visible result. If your ladder is not visible as well as your experimental lanes, that suggests problems with transfer or hybridization. Adjust your hybridization temperature lower if your signal is weak or higher if it is overexposed. Make sure your transfers are not being short-circuited; surround the edge of your gel/blot stack with parafilm or plastic wrap. Finally, look at your wash steps after hybridization; make sure you are not washing the blot too much or too little. Adjust wash temperatures and wash formulations as needed; these can be impacted by the probe sequence and its homology to the sequence being probed.
Southerns are tricky because there are a lot of places where the process can go wrong, so it will take some effort and patience to figure it out. Test out different conditions methodically and find which offers the best solution. Best of luck.
- Badges
- Science method